AuC-containing materials, their preparation and use

Abstract

To provide a gold cluster-containing substance having pharmaceutical activity, the substance comprising a gold cluster and a ligand Y that covers the outside of the gold cluster.SOLUTION: A pharmaceutical use of a gold cluster and a gold cluster-containing substance, as well as a method for manufacturing and using the same, are disclosed. The gold cluster and the gold cluster-containing substance as described above can inhibit the aggregation of Aβ and α-syn, and have excellent effects at levels of a cell model and an animal model, and can be used for producing a drug for preventing and treating Alzheimer's disease and / or Parkinson's disease.SELECTED DRAWING: Figure 12

Description

[Technical Field]

[0001] The present invention relates to the technical field of nanometer nanomedicine, in particular to a gold cluster (AuC)-containing material, as well as its preparation method and use. [Background technology]

[0002] Neurodegenerative diseases are a major threat to human health. Their common pathological features are abnormal protein entanglement and its amyloidosis in neurons, as well as associated neuronal apoptosis and neurological disorders. Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common. The clinical signs of AD are characterized by memory and cognitive dysfunction and personality and behavioral changes, while the clinical signs of PD mainly include tremor, bradykinesia, muscle rigidity, orthostatic gait disturbance, and other movement disorders. Both AD and PD primarily occur in elderly people, and their prevalence increases with age. For example, the prevalence of AD is 5% among people over 65 years of age, but exceeds 30% among people over 80 years of age. Therefore, the number of patients suffering from these two diseases is constantly increasing as life expectancy increases and the aging of the population increases. Alzheimer's disease (AD), by far, affects more than 40 million people, a figure that is expected to reach 150 million by 2050. In the United States alone, over 200 billion US dollars—twice as much as cancer—is spent annually on treating AD patients, making it the world's most expensive disease. The number of PD patients worldwide is conservatively estimated to exceed 10 million. However, the etiology of these two diseases remains unknown. Regarding clinical treatment, several drugs have been approved by the U.S. FDA for the treatment of mild and moderate AD or PD. However, these drugs are neurotransmitter modulators that can only temporarily improve patients' cognitive or motor function. Discontinuing these drugs quickly leads to reversal of symptoms. To date, no drugs exist that can terminate or reverse the pathological processes of these two diseases. Therefore, developing new drugs for the treatment of AD or PD is highly significant.

[0003] Research has shown that amyloid proteins in the brains of AD patients are primarily beta-amyloid (Aβ) and tau proteins, with small amounts of alpha-synuclein (α-syn), and that the initial site of onset is the hippocampus, which plays a role in memory, learning, and spatial orientation. Brain damage in PD patients begins in the substantia nigra, which plays a role in somatomotor function. The difference in the initial site of onset determines the different symptoms of patients with these two diseases. However, research has shown that more than half of AD patients develop motor disorders in the later stages, and most PD patients also share the same symptoms as AD patients in the later stages. These phenomena suggest that the two diseases have an inherent correlation in their etiology and progression.

[0004] The formation of senile plaques in the brain is one of the fundamental pathological characteristics of AD. Aβ, a major component of senile plaques, is a polypeptide consisting of 36 to 43 amino acids. This polypeptide is a hydrolysis product of amyloid precursor protein (APP), with Aβ(1-40) accounting for more than 90% of the total Aβ content. Recent studies have revealed that Aβ has normal physiological functions and can regulate intersynaptic acetylcholinergic signaling by regulating the catalytic activity of cholinesterase. However, excessive Aβ aggregation and fibrillation in the brain can cause synaptic dysfunction and subsequent secondary inflammatory responses, leading to the loss of neuronal function and neuronal death. Therefore, the development of substances that can inhibit Aβ aggregation and fibrillation and block its neurotoxicity is an important approach for the research and development of AD drugs.

[0005] The pathological hallmark of PD is the progressive loss of dopaminergic (DA) neurons, primarily in the nigrostriatal system, accompanied by the formation of Lewy bodies. Lewy bodies contain hollow, radial amyloid fibrils formed primarily by the aggregation of denatured α-syn. α-syn is located at the presynaptic membrane terminals of neurons, and its natural state within these bodies is soluble and unfolded. Under pathological conditions, α-syn misfolds, resulting in the formation of β-sheet structures, which then undergo further aggregation and fibrillation to form Lewy body lesions. Research has indicated that α-syn amyloidosis plays a key role in the pathological process of the disease. Therefore, inhibiting α-syn aggregation and fibrillation has become one approach in the research and development of drugs for the prevention and treatment of PD. On the other hand, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin. MPTP itself is not toxic, but after entering the brain, its metabolism produces 1-methyl-4-phenylpyridine cation (MPP + ) can destroy DAergic neurons in the substantia nigra. + MPTP and MPP can interfere with NADH dehydrogenase, a key component in the respiratory chain of mitochondrial metabolism, leading to cell death and the accumulation of free radicals. The massive death of DAergic neurons caused by this process has a significant impact on motor control in the cerebral cortex, resulting in symptoms similar to those of PD. + It is widely used in establishing PD-related animal and cell models, as well as in the research and development of PD drugs.

[0006] Gold nanoparticles are nanoscale gold particles (the diameter of the gold core of gold nanoparticles used in research is generally greater than 3 nm). Due to their unique optical and electrical properties, good biocompatibility, and easy surface modification, gold nanoparticles are widely used in biology and related medical fields, such as biosensors, medical imaging, and tumor detection. Due to their chemical inertness, large specific surface area, and ability to penetrate the blood-brain barrier at low concentrations, gold nanoparticles are also used as drug carriers in research into directional drug delivery and controlled release. In recent years, studies have been conducted on the conjugation of gold nanoparticles with specific ligands (e.g., heteropolyacids and polypeptides with specific sequences) that inhibit the aggregation of fibrillar proteins, and have shown some efficacy in in vitro protein fibrillation inhibition experiments (Non-Patent Documents 1, 2, and 3). However, cell model results have shown that gold nanoparticles (gold core size greater than 5 nm) exhibit a synergistic effect on cell viability when used together with compounds that have a protective effect against fibrin-damaged cells (Non-Patent Document 4), but the effect is not clear when they are used alone. No AD experiments at the level of animal models have been reported yet. Moreover, in these studies, gold nanoparticles were primarily used as drug carriers rather than as active ingredients.

[0007] Gold clusters (AuC) are ultrafine gold nanoparticles with a gold core diameter of less than 3 nm. They contain only a few to several hundred gold atoms, which disrupts the face-centered cubic packing of gold atoms in conventional gold nanoparticles and splits their energy levels, resulting in molecular-like properties that are completely different from conventional gold nanoparticles larger than 3 nm. Due to the energy level splitting, AuC exhibits excellent fluorescence emission properties similar to those of semiconductor quantum dots, without the surface plasmon effect and optical properties of conventional gold nanoparticles. Furthermore, in the UV-visible absorption spectrum of AuC, the plasmon resonance peak at 520 ± 20 nm disappears, while one or more new absorption peaks appear above 560 nm, which are not observed in conventional gold nanoparticles. Therefore, the disappearance of the plasmon resonance absorption peak (520 ± 20 nm) and the appearance of new absorption peaks above 560 nm in the UV-visible absorption spectrum are important indicators for determining whether AuC has been successfully produced (Non-Patent Document 5). AuC also possesses magnetic, electrical, and catalytic properties, as well as photothermal effects, that are significantly different from those of conventional gold nanoparticles, making AuC potentially useful in the fields of single-molecule optoelectronics, molecular catalysis, and photothermal conversion. It has wide application prospects.

[0008] Furthermore, AuC is also used in the fields of bioprobes and medical imaging due to its excellent fluorescence properties. For example, Sandeep Verma's team has Purine-modified AuC has been used as a green fluorescent probe for imaging (Non-Patent Document 6). This type of document utilizes the fluorescent properties of AuC and does not require the pharmaceutical activity of AuC itself. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Y. H. Liao, Y. J. Chang, Y. Yoshiike, Y. C. Chang, Y. R. Chen, Small 2012, 8, 3631 [Non-Patent Document 2] Y. D. Alvarez, J. A. Fauerbach, J. V. Pellegrotti, T. M. Jovin, E. A. Jares-Erijman, F. D. Stefani, Nano Letters 2013, 13, 6156 [Non-Patent Document 3] S. Hsieh, C. W. Chang, H. H. Chou, Colloids and Surfaces B: Biointerfaces, 2013, 112, 525 [Non-Patent Document 4] N. Gao, H. Sun, K. Dong, J. Ren, X. Qu, Chemistry-A European Journal 2015, 21, 829 [Non-Patent Document 5] H.F. Qian, M.Z. Zhu, Z.K. Wu, R.C. Jin, Accounts of Chemical Research 2012, 45, 1470 [Non-Patent Document 6] J. R. Wallbank, D. Ghazaryan, A. Misra, Y. Cao, J. S. Tu, B. A. Piot, M. Potemski, S.Wiedmann, U. Zeitler, T. L. M. Lane, S.V. Morozov, M. T. Greenaway, L. Evaes, A. K. Geim, V. I. Falko, K. S. Novoselov, A. Mishchenko, ACS Applied Materials & Interfaces 2014, 6, 2185 [Summary of the Invention] [Problems to be Solved by the Invention]

[0010] SUMMARY OF THE INVENTION It is an object of the present invention to address the technical disadvantages in the prior art. [Means for solving the problem]

[0011] In a first aspect, the present invention provides a pharmaceutically active AuC-containing material comprising AuC and a ligand Y coating the outside of the AuC.

[0012] The gold core diameter of the AuC is less than 3 nm, preferably 0.5 nm to 2.6 nm.

[0013] Ligand Y includes, but is not limited to, one or more of L(D)-cysteine ​​and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0014] The L(D)-cysteine ​​and its derivatives are preferably L(D)-cysteine, N-isobutyryl-L(D)-cysteine ​​(L(D)-NIBC), or N-acetyl-L(D)-cysteine ​​(L(D)-NAC).

[0015] The cysteine-containing oligopeptides and derivatives thereof are preferably cysteine-containing dipeptides, cysteine-containing tripeptides, or cysteine-containing tetrapeptides.

[0016] The cysteine-containing dipeptide is preferably L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), L-histidine-L-cysteine ​​dipeptide (HC), or L-cysteine-L-histidine dipeptide (CH).

[0017] The cysteine-containing tripeptide is preferably glycine-L-cysteine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR), L-lysine-L-cysteine-L-proline tripeptide (KCP), or L-glutathione (GSH).

[0018] The cysteine-containing tetrapeptide is preferably glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) or glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR).

[0019] The other thiol-containing compound is preferably 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trolobol, N-(2-mercaptopropionyl)-glycine, or dodecyl mercaptan.

[0020] The material is in powder or flocculent form.

[0021] In a second aspect, the present invention provides a method for producing an AuC-containing material, comprising the steps of: (1) dissolving HAuCl4 in one of methanol, water, ethanol, n-propanol, and ethyl acetate to obtain a solution A having a HAuCl4 concentration of 0.01M to 0.03M; (2) dissolving a ligand Y in a solvent to obtain a solution B having a ligand Y concentration of 0.01 M to 0.18 M; (3) mixing solution A in step (1) and solution B in step (2) at a molar ratio between HAuCl4 and ligand Y of 1:(0.01-100) (preferably 1:(0.1-10), more preferably 1:(1-10)), stirring them in an ice bath for 0.1 to 48 hours (preferably 0.1 to 24 hours, more preferably 0.5 to 2 hours), adding a 0.025M to 0.8M NaBH4 solution (preferably an aqueous solution of NaBH4, an ethanol solution of NaBH4, or a methanol solution of NaBH4), and then continuing to stir in an ice-water bath for 0.1 to 12 hours (preferably 0.1 to 2 hours, more preferably 1 to 2 hours) at a molar ratio between NaBH4 and ligand Y of 1:(0.01-100) (preferably 1:(0.1-8), more preferably 1:(1-8)); (4) centrifuging the reaction solution in step (3) at 8000 rpm to 17500 rpm for 10 minutes to 100 minutes to obtain AuC precipitates with various average particle sizes, preferably using an ultrafiltration tube with a MWCO of 3K to 30K to centrifugate the reaction solution in step (3) at 8000 rpm to 17500 rpm for 10 minutes to 100 minutes using a gradient to obtain AuC with various average particle sizes; (5) dissolving the AuC precipitates of various average particle sizes obtained in step (4) in water, placing the solution in a dialysis bag, and dialyzing the solution in water at room temperature for 1 to 7 days; (6) freeze-drying the AuC solution in the dialysis bag for 12 to 24 hours to obtain an AuC-containing substance; The present invention provides a method comprising:

[0022] The solvent in step (2) is one or more of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, ethanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl acetate, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, and propyl acetate.

[0023] In a third aspect, the present invention provides the use of AuC-containing materials in near-infrared fluorescent probes in the fields of catalyst production or molecular catalysis, chiral recognition, molecular detection, biomedical detection, and imaging.

[0024] In a fourth aspect, the present invention provides use of the AuC-containing material in the manufacture of a medicament for a disease associated with Aβ aggregation and fibrillation, and a disease associated with α-syn aggregation and fibrillation.

[0025] In a fifth aspect, the present invention provides the use of an AuC-containing material in the manufacture of a medicament for the prevention and treatment of AD.

[0026] In a sixth aspect, the present invention provides the use of an AuC-containing material in the manufacture of a medicament for the prevention and treatment of PD.

[0027] In a seventh aspect, the present invention provides the use of AuC in the manufacture of a medicament for a disease associated with Aβ aggregation and fibrillization.

[0028] The disease associated with the aggregation and fibrillation of Aβ is AD.

[0029] The AuCs were modified with L-glutathione (GSH), N-acetyl-L(D)-cysteine ​​(L(D)-NAC), N-isobutyryl-L(D)-cysteine ​​(L(D)-NIBC), L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap), or L(D)-cysteine ​​(L(D)-Cys).

[0030] In an eighth aspect, the present invention provides the use of AuC in the manufacture of a medicament for a disease associated with α-syn aggregation and fibrillization.

[0031] The disease associated with the aggregation and fibrillation of Aβ is PD.

[0032] The AuCs were modified with L-glutathione (GSH), N-acetyl-L(D)-cysteine ​​(L(D)-NAC), N-isobutyryl-L(D)-cysteine ​​(L(D)-NIBC), L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap), or L(D)-cysteine ​​(L(D)-Cys).

[0033] The AuC-containing substance provided by the present invention exhibits excellent effects on inhibiting the aggregation of Aβ and α-syn in in vitro experiments for the inhibition of Aβ and α-syn aggregation, and is effective in Aβ-induced cellular AD models and MPP. + In experiments with an induced cell PD model, the AuC-containing substance showed excellent effects in improving cell viability. In a transgenic mouse model of AD, the AuC-containing substance can significantly improve the cognitive behavioral performance of diseased mice and play a major role in inhibiting the formation of Aβ(1-40) plaques and Aβ(1-42) plaques in the mouse hippocampus and cerebral cortex. In an MPTP-induced PD mouse model, the AuC-containing substance significantly improved and cured the dyskinetic disorders of MPTP-lesioned mouse models, improved the motor performance of diseased mice, and essentially inhibited MPTP-induced specific apoptosis of DAergic neurons in the substantia nigra and striatum of mice. Furthermore, the AuC-containing substance has good biosafety at both the cellular and animal levels. The above results indicate that the AuC-containing substance provided by the present invention not only affects the aggregation and fibrillation of fibrous proteins, but also affects the process of neurodegenerative diseases at a deeper level, for example, signal transduction functions related to neuronal energy metabolism and neurotransmitter metabolism. Therefore, the AuC-containing substance provided by the present invention is important for the research and development of new medicines for neurodegenerative diseases, such as AD and / or PD.

[0034] On the other hand, the ligand molecule did not show any inhibitory effect in the kinetics experiment for in vitro inhibition of Aβ aggregation and in the Aβ lesion AD cell model test, or showed no inhibitory effect in the Aβ lesion AD cell model and MPP + Since no increase in cell viability was observed in the diseased PD cell model, it is suggested that the efficacy in AD and PD is due to AuC rather than the ligand. Based on the pharmaceutical activity of AuC itself, the development of new competitive drugs is expected. [Brief explanation of the drawings]

[0035] [Figure 1]1 shows ultraviolet-visible (UV) spectra, transmission electron microscope (TEM) images, and particle size distribution diagrams of ligand L-NIBC modified gold nanoparticles with various particle sizes. [Figure 2] Figure 1 shows UV-visible spectra, TEM images, and particle size distribution diagrams of ligand L-NIBC modified AuC with various particle sizes. [Figure 3] 1 shows the infrared spectra of ligand L-NIBC modified AuC with different particle sizes. [Figure 4] AFM topography of Aβ(1-40) after incubation with ligand L-NIBC modified gold nanoparticles or AuC for 48 hours. [Figure 5] 1 shows the kinetic curves of Aβ fibrillization of gold nanoparticles and AuC modified with the ligand L-NIBC at various particle sizes and various concentrations. [Figure 6] FIG. 1 shows a diagram illustrating the effect of different particle sizes and different concentrations of ligand L-NIBC modified gold nanoparticles or AuC on cell viability in an Aβ-induced AD cell model. [Figure 7] 1 shows UV, infrared, TEM, and particle size distribution diagrams of AuC modified with the ligand CR (CR-AuC). [Figure 8] UV, infrared, TEM, and particle size distribution diagrams of AuC modified with the ligand RC (RC-AuC) are shown. [Figure 9] 1 shows UV, infrared, TEM, and particle size distribution diagrams of AuC modified with 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e., Cap). [Figure 10] 1 shows UV, infrared, TEM, and particle size distribution diagrams of AuC modified with the ligand GSH (GSH-AuC). [Figure 11] 1 shows UV, infrared, TEM, and particle size distribution diagrams of AuC modified with the ligand D-NIBC (D-NIBC-AuC). [Figure 12] 1 shows the inhibitory effect curves of AuC modified with various ligands on the aggregation and fibrillization of Aβ(1-40). [Figure 13]10 shows a schematic diagram of a water maze experimental device in embodiment 5. FIG. [Figure 14] FIG. 1 shows a diagram showing the effect of an AuC-containing substance on cognitive behavior in an APP / PS1 double transgenic C57BL / 6 mouse model (150 days after administration). [Figure 15] FIG. 1 shows a diagram showing the effect of an AuC-containing substance on the expression of Aβ(1-40) in the hippocampus and cerebral cortex of mice in an APP / PS1 double transgenic C57BL / 6 mouse model (100 days after administration). [Figure 16] FIG. 1 shows a diagram showing the effect of an AuC-containing substance on the expression of Aβ(1-42) in the hippocampus and cerebral cortex of mice in an APP / PS1 double transgenic C57BL / 6 mouse model (100 days after administration). [Figure 17] FIG. 1 shows a diagram showing the effect of an AuC-containing substance on the expression of Aβ(1-40) in the hippocampus and cerebral cortex of mice in an APP / PS1 double transgenic C57BL / 6 mouse model (150 days after administration). [Figure 18] FIG. 1 shows a diagram showing the effect of an AuC-containing substance on the expression of Aβ(1-42) in the hippocampus and cerebral cortex of mice in an APP / PS1 double transgenic C57BL / 6 mouse model (150 days after administration). [Figure 19] FIG. 1 shows the effect of AuC-containing substances on the kinetics of α-syn fibrillization. [Figure 20] FIG. 1 shows a diagram illustrating the effect of AuC-containing substances on cell viability in an MPP+lesioned PD cell (SH-sy5y) model. [Figure 21] FIG. 1 shows a diagram illustrating the effect of AuC-containing substances on cell apoptosis in an MPP+-induced PD cell (PC12) model. [Figure 22] FIG. 1 shows a diagram showing the effect of an AuC-containing substance on the spontaneous movement of MPTP-lesioned model mice. [Figure 23] FIG. 1 shows a diagram showing the effect of an AuC-containing substance on the swimming ability of MPTP-lesioned model mice. [Figure 24]FIG. 1 shows a diagram illustrating the effect of an AuC-containing substance on rotarod behavior in MPTP-lesioned model mice. [Figure 25] FIG. 1 shows a diagram illustrating the effect of an AuC-containing substance on DAergic neurons in the substantia nigra and striatum of MPTP-lesioned model mice. [Figure 26] FIG. 1 shows a diagram illustrating the effect of AuC-containing materials of different particle sizes and different concentrations on SH-sy5y neuroblastoma cell viability. DETAILED DESCRIPTION OF THE INVENTION

[0036] By studying the effect of gold nanoparticles with a certain ligand on Aβ aggregation, we found that when the gold core diameter of gold nanoparticles was changed from large to small, the promoting effect of gold nanoparticles with the same ligand on Aβ aggregation switched to an inhibitory effect, and complete inhibition of Aβ aggregation could be achieved when the particle size was small enough to be AuC. Furthermore, we found that AuC has a complete inhibitory effect on α-syn. In this effect, AuC itself, not the ligand, plays the inhibitory role.

[0037] Generally, the gold core diameter of the gold nanoparticles used in the above studies is larger than 3 nm; when the gold core diameter is smaller than 3 nm, the gold nanoparticles are called AuC. The disappearance of the plasmon resonance absorption peak (520±20 nm) and the appearance of a new absorption peak above 560 nm in the UV-visible absorption spectrum indicate the successful preparation of AuC. Without a ligand, AuC cannot exist stably in solution. When AuC binds to a thiol-containing ligand, ligand-modified AuC (also called AuC) is formed via an Au-S bond.

[0038] Existing ligand-modified AuCs disclosed in the literature include those modified with L-glutathione (GSH), N-acetyl-L(D)-cysteine ​​(L(D)-NAC), N-isobutyryl-L(D)-cysteine ​​(L(D)-NIBC), etc. Preparation methods are shown in the literature (HF Qian, MZ Zhu, ZK Wu, RC Jin, Accounts of Chemical Research 2012, 45, 1470; C. Gautier, T. Buergi, Journal of the American Chemical Society 2006, 128, 11079), and they are mainly used for catalysis, chiral recognition, molecular biology, and so on. It has applications in the fields of detection, biosensing, drug delivery, and bioimaging (G. Li, RC Jin, Accounts of Chemical Research 2013, 46, 1749, HF Qian, M. Z. Zhu, ZK Wu, RC Jin, Accounts of Chemical Research 2012, 45, 1470, JF Parker, CA Fields-Zinna, RW Murray, Accounts of Chemical Research 2010, 43, 1289, SH Yau, O. Varnavski, T. Goodson, Accounts of Chemical Research 201 3, 46, 1506).

[0039] In the present invention, the effects of AuC on AD and / or PD are investigated, which initially involves at least using AuC of various sizes containing various ligands (ligands that do not have an inhibitory effect on Aβ aggregation) as study subjects. In vitro experiments on the inhibition of Aβ aggregation and α-syn aggregation, and Aβ-induced AD cell models and MPPs are also performed. +Through three-stage experimental studies, including experiments on an induced PD cell model, experiments on an AD transgenic mouse model, and experiments on an MPTP-induced PD mouse model, and also through acute toxicity experiments in mice considering AuC cytotoxicity, in vivo distribution experiments in mice, etc., ligand-modified AuCs were provided and their use in the manufacture of drugs to treat AD and PD was found. Comparing these results with the experimental results on gold nanoparticles, it was shown that gold nanoparticles with a diameter larger than 3 nm do not have the desired effect for this purpose and cannot be used to manufacture drugs to treat AD or PD, while ligand-modified AuCs can be used to manufacture drugs to treat AD and / or PD.

[0040] The present invention is further described in the following embodiments, which should not be construed as limiting the present invention in any way.

[0041] The purity of the raw materials used in the following embodiments is chemical purity or higher, and they can all be purchased from the market.

[0042] Example 1: Preparation of ligand-modified AuC This embodiment provides a method for producing a ligand-modified AuC, comprising the steps of: (1) dissolving HAuCl4 in one of methanol, water, ethanol, n-propanol, and ethyl acetate to obtain a solution A having a HAuCl4 concentration of 0.01M to 0.03M; (2) dissolving a ligand Y in a solvent to obtain a solution B having a ligand Y concentration of 0.01 M to 0.18 M; Ligand Y includes, but is not limited to, L(D)-cysteine ​​and other cysteine ​​derivatives, such as N-isobutyryl-L-cysteine ​​(L-NIBC), N-isobutyryl-D-cysteine ​​(D-NIBC), N-acetyl-L-cysteine ​​and N-acetyl-D-cysteine, cysteine-containing oligopeptides and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides and other cysteine-containing peptides, such as L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine ​​dipeptide (RC), L-cysteine-L-histidine (CH), glycine-L-cysteine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR), L-glutathione (GSH), glycine-L-serine ... and one or more of glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GSCR) and glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR) and other thiol-containing compounds, such as 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-trolobol, and dodecyl mercaptan, in a solvent selected from the group consisting of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, ethanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl acetate, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, and propyl acetate; (3) Solution A and solution B were mixed together in a solution containing HAuCl4 and ligand Y in a molar ratio of 1:(0.01 to 100), stirring them in an ice bath for 0.1 to 48 hours, adding a 0.025M to 0.8M aqueous, ethanol, or methanol solution of NaBH4, and continuing to stir in an ice-water bath to react for 0.1 to 12 hours; The molar ratio between NaBH4 and ligand Y is 1:(0.01~100); (4) Using an ultrafiltration tube with a MWCO of 3K to 30K, the reaction solution is centrifuged at 8,000 to 17,500 rpm for 10 to 100 minutes after the completion of the reaction, thereby obtaining ligand-modified AuC precipitates with various average particle sizes (the specific gradient centrifugation is as described in (4) of embodiment 2. The opening of the filtration membrane for ultrafiltration tubes with various MWCOs directly determines the size of AuC that can pass through the membrane); This step can be omitted. In other words, after completing step (3), step (5) can be started directly to obtain mixed AuCs with various sizes; (5) dissolving the AuC precipitates of various average particle sizes obtained in step (4) in water, placing the solution in a dialysis bag, and dialyzing the solution in water at room temperature for 1 to 7 days; (6) freeze-drying the dialyzed AuC for 12 to 24 hours to obtain a powder or flocculent material, i.e., ligand-modified AuC; The present invention discloses a method including:

[0043] As detected (specific detection methods are provided in Example 2), the particle size of the powder or flocculant obtained by the above method is less than 3 nm (generally ranging from 0.5 nm to 2.6 nm). The UV-visible absorption spectrum has one or more absorption peaks above 560 nm and no obvious absorption peak at 520 nm. This confirms that the powder or flocculant obtained is AuC.

[0044] Example 2: Preparation and characterization of AuC modified with various ligands Taking the ligand L-NIBC as an example, the preparation and characterization of AuC modified with the ligand L-NIBC will be described in detail. (1) Weigh out 1.00 g of HAuCl4 and dissolve it in 100 mL of methanol to obtain a 0.03 M solution A. (2) Weigh out 0.57 g of L-NIBC and dissolve it in 100 mL of glacial acetic acid (acetic acid) to obtain a 0.03 M solution B. (3) 1 mL of solution A was weighed and mixed with 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of solution B (i.e., the molar ratios between HAuCl4 and L-NIBC were 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively), and the mixture was reacted for 2 hours with stirring in an ice bath. When the solution turned from golden yellow to colorless, 1 mL of freshly prepared 0.03 M aqueous NaBH4 solution (prepared by weighing 11.3 mg of NaBH4 and dissolving it in 10 mL of ethanol) was quickly added. After the solution turned dark brown, the reaction was continued for 30 minutes, and then 10 mL of acetone was added to stop the reaction. (4) After the reaction, the reaction solution was subjected to gradient centrifugation to obtain L-NIBC-modified AuC powders with various particle sizes. Specific method: After the reaction was completed, the reaction solution was transferred to a 50 mL ultrafiltration tube with a MWCO of 30K and centrifuged at 10,000 rpm for 20 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of approximately 2.6 nm. The mixed solution in the outer tube was then transferred to a 50 mL ultrafiltration tube with a MWCO of 10K and centrifuged at 13,000 rpm for 30 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of approximately 1.8 nm. The mixed solution in the outer tube was then transferred to a 50 mL ultrafiltration tube with a MWCO of 3K and centrifuged at 17,500 rpm for 40 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of approximately 1.1 nm. can be. (5) The powders of three different particle sizes obtained by gradient centrifugation are sedimented, the solvent is removed, and the crude product is air-dried with N2, dissolved in 5 mL of ultrapure water, placed in a dialysis bag (MWCO is 3 KDa), placed in 2 L of ultrapure water, and dialyzed for 7 days with water change every other day, freeze-dried, and stored for later use.

[0045] Characterization experiments were carried out on the powder obtained above (AuC modified with the ligand L-NIBC). Meanwhile, gold nanoparticles modified with the ligand L-NIBC were used as a control. The preparation method of gold nanoparticles with the ligand L-NIBC was described in reference (W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and N.A. Kotov, Journal of the American Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo, Q. Yao, D.T. Leong, N. Yan and J. Xie, Angewandte Chemie International Edition 2014, 53, 4623).

[0046] 1. Morphological observation by transmission electron microscope (TEM) The test powders (the L-NIBC-modified AuC sample and the L-NIBC-modified gold nanoparticle sample prepared in Example 2) were dissolved in ultrapure water to a concentration of 2 mg / L. Test samples were then prepared using the hanging drop method. Specific method: 5 μL of the sample was dropped onto an ultrathin carbon film and allowed to evaporate until the water droplets disappeared. The morphology of the sample was then observed using a JEM-2100F STEM / EDS field emission high-resolution TEM.

[0047] Four TEM images of ligand L-NIBC-modified gold nanoparticles are shown in Panels B, E, H, and K of Figure 1, and three TEM images of ligand L-NIBC-modified AuC are shown in Panels B, E, and H of Figure 2.

[0048] The images in Figure 2 show that the L-NIBC-modified AuC samples have uniform particle size and good dispersibility, and the average diameters (referring to the diameter of the gold core) of the L-NIBC-modified AuC are 1.1 nm, 1.8 nm, and 2.6 nm, respectively, which is in good agreement with the results in Panels C, F, and I of Figure 2. In comparison, the gold nanoparticle samples modified with the ligand L-NIBC have larger particle size. Their average diameters (referring to the diameter of the gold core) are 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively, which is in good agreement with the results in Panels C, F, I, and L of Figure 1.

[0049] 2. Ultraviolet (UV)-visible (vis) absorption spectrum The test powder was diluted to a concentration of 10 mg L -1 The sample was dissolved in ultrapure water until the concentration reached 100 ppm and measured by UV-vis absorption spectroscopy at room temperature. The scan range was 190 nm to 1100 nm. The sample cell was a standard quartz cuvette with a 1 cm optical path, and the reference cell was filled with ultrapure water.

[0050] The UV-vis absorption spectra of four ligand L-NIBC-modified gold nanoparticle samples with different sizes are shown in Panels A, D, G, and J of Figure 1, and the statistical distribution of particle sizes is shown in Panels C, F, I, and L of Figure 1. The UV-vis absorption spectra of three ligand L-NIBC-modified AuC samples with different sizes are shown in Panels A, D, and G of Figure 2, and the statistical distribution of particle sizes is shown in Panels C, F, and I of Figure 2.

[0051] Figure 1 shows that gold nanoparticles modified with the ligand L-NIBC are The results show that the UV absorption peak was at about 520 nm. The position of the absorption peak is related to the particle size. When the particle size is 3.6 nm, the UV absorption peak appears at 516 nm, when the particle size is 6.0 nm, the UV absorption peak appears at 517 nm, when the particle size is 10.1 nm, the UV absorption peak appears at 520 nm, and when the particle size is 18.2 nm, the absorption peak appears at 523 nm. None of the four samples has any absorption peaks above 560 nm.

[0052] Figure 2 shows that the UV absorption spectra of the three L-NIBC-modified AuC samples with different particle sizes in Example 2 show that the surface plasmon absorption peak at 520 nm disappears, and two distinct absorption peaks appear above 560 nm. The positions of the absorption peaks change slightly with the particle size of the AuC. This is because AuC exhibits molecular-like properties due to the collapse of its face-centered cubic structure, which leads to discontinuities in the density of states of AuC, splitting the energy levels, eliminating the plasmon resonance effect, and introducing new absorption peaks toward longer wavelengths. It can be concluded that the three powder samples with different particle sizes obtained in Example 2 are all ligand-modified AuC.

[0053] 3. Fourier transform infrared spectroscopy Infrared spectra were measured on a VERTEX80V Fourier transform infrared spectrometer manufactured by Bruker in the high vacuum total reflection mode of solid powders. The scan range was 4000 cm. -1 ~400cm -1 The number of scans was 64. Taking the L-NIBC-modified AuC sample prepared in Example 2 as an example, the test samples were L-NIBC-modified AuC dry powders with three different particle sizes, and the control sample was pure L-NIBC powder. The results are shown in Figure 3.

[0054] Figure 3 shows the infrared spectra of L-NIBC-modified AuC with various particle sizes. Compared with pure L-NIBC (top curve), all of the SH stretching vibrations of L-NIBC-modified AuC with various particle sizes are at 2500 cm. -1 ~2600cm -1 The infrared spectrum of the ligand-modified AuC completely disappeared at 1000 nm, while other characteristic peaks of L-NIBC were still observed, indicating that the L-NIBC molecules were successfully tethered to the surface of AuC via Au-S bonds. The figure also shows that the infrared spectrum of the ligand-modified AuC is independent of its size.

[0055] AuC modified with other ligands Y were prepared by the same method as above, except that the solvent of solution B, the feed ratio between HAuCl4 and ligand Y, the reaction time, and the amount of NaBH4 added were slightly adjusted. For example, when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC), or N-isobutyryl-D-cysteine ​​(D-NIBC) was used as ligand Y, acetic acid was selected as the solvent; when dipeptide CR, dipeptide RC, or 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline was used as ligand Y, water was selected as the solvent, etc. Since the other steps were similar, they will not be described in detail herein.

[0056] In the present invention, a series of ligand-modified AuCs were prepared and obtained by the above method. The ligands and preparation method parameters are listed in Table 1.

[0057] [Table 1]

[0058] The samples in the embodiments listed in Table 1 are confirmed by the above method. Figures 7-11 show UV spectra (panels A in Figures 7-11), infrared spectra (panels B in Figures 7-11), transmission electron microscope (TEM) images (panels C in Figures 7-11), and particle size distributions (panels D in Figures 7-11) of AuC modified with the ligands CR, RC, 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (abbreviation: Cap), GSH, and D-NIBC.

[0059] The results show that the diameters of AuC modified with various ligands from Table 1 are all smaller than 3 nm. The UV spectra also show a peak at 520±20 nm. The Fourier transform infrared spectra also show the disappearance of the IR absorption peak of the ligand thiol (between the dotted lines in panel B of Figures 7 to 11) while all other characteristic infrared peaks are maintained, suggesting that all the ligand molecules are successfully tethered to the surface of AuC and that the present invention has successfully obtained AuC modified with the ligands listed in Table 1.

[0060] Embodiment 3: In vitro Aβ aggregation kinetics experiment This embodiment confirmed the functionality of the ligand-modified AuC through in vitro experiments of Aβ aggregation kinetics, and compared the effects of ligand-modified gold nanoparticles and the ligand molecule independently on Aβ aggregation kinetics, demonstrating that the functionality was due to AuC and not the ligand. In these experiments, ThT fluorescent labeling was used to characterize the aggregation and fibrillization kinetics of Aβ(1-40).

[0061] Thioflavin T (ThT) is a dye specifically staining amyloid fibrils. When ThT is incubated with polypeptide or protein monomers, its fluorescence remains almost unchanged. When ThT encounters amyloid polypeptides or proteins with fibrillar structures, it immediately binds to the amyloid polypeptides or proteins, resulting in an exponential increase in its fluorescence intensity. Precisely due to this property, ThT is widely used as a marker for monitoring peptide or protein amyloidosis. The fibrillization process of Aβ(1-40) is also a polymerization process controlled by nucleation. Therefore, the growth curve of Aβ(1-40) fibrils measured by the ThT fluorescent labeling method can be divided into three main phases: the initial phase, the growth phase, and the platform phase. The initial phase is primarily a phase in which Aβ(1-40) undergoes conformational transitions to form misfolded structures, followed by aggregation and nucleation. The growth stage is when Aβ(1-40) monomers accumulate axially on the oligomer core, forming fibrils and growing rapidly. The platform stage is when all Aβ(1-40) molecules have formed complete, long fibrils, meaning that fibrils no longer grow. ThT fluorescent labeling can easily monitor the dynamics of Aβ(1-40) fibrillar aggregation.

[0062] 1) Pretreatment of Aβ(1-40) monomers Lyophilized powder of amyloid polypeptide Aβ(1-40) (Invitrogen Corp.) was dissolved in hexafluoroisopropanol (HFIP) to obtain a 1 g / L Aβ(1-40) solution. The solution was sealed and incubated at room temperature for 2 to 4 hours. The HFIP was then air-dried (approximately 1 hour) in a fume hood using high-purity nitrogen (N2, 99.9%) at an appropriate flow rate. Finally, the dried Aβ(1-40) was dissolved in 200 μL of DMSO. After sealing, the solution was kept at -20°C in a refrigerator for up to 1 week for further use. Before use, the DMSO solution of amyloid polypeptide was diluted with a large amount of phosphate buffer solution (PBS, 10 mM, pH = 7.4) until the Aβ(1-40) concentration reached 20 μM, thereby obtaining an Aβ(1-40) PBS solution. All Aβ(1-40) PBS solutions for experiments were freshly prepared.

[0063] 2) Sample preparation and detection Ligand-modified AuC and gold nanoparticles were added to 20 μM Aβ(1-40) PBS to form AuC samples with various concentrations and particle sizes, and correspondingly, gold nanoparticle samples with various ligands. These samples were continuously incubated in a 96-well plate at 37 °C using ThT fluorescent labeling, and the fluorescence intensity was monitored every 10 min using a microplate reader. The kinetics of Aβ(1-40) aggregation were characterized by changes in ThT fluorescence intensity. Evaluated.

[0064] Three sizes of L-NIBC-modified AuC, with particle sizes of 2.6 nm, 1.8 nm, and 1.1 nm, respectively, prepared in Example 2, were used as experimental groups. Four sizes of L-NIBC-modified gold nanoparticles, with particle sizes of 18.2 nm, 10.1 nm, 6.0 nm, and 3.6 nm, respectively, and L-NIBC molecules not bound to AuC or gold nanoparticles were used as controls. All sizes of AuC or gold nanoparticles were present at six concentrations: 0 ppm (blank control containing no AuC, gold nanoparticles, or L-NIBC), 0.1 ppm, 1.0 ppm, 5.0 ppm, 10.0 ppm, and 20.0 ppm, respectively. L-NIBC molecules were used at two concentrations: 1.0 ppm and 10.0 ppm, respectively.

[0065] The results are shown in FIGS.

[0066] Figure 4 shows AFM topographies of Aβ(1-40) after co-incubation for 48 hours with each experimental group and control group. Panel A shows the AFM topographies of Aβ(1-40) after 48 hours of incubation alone. Panel B shows the AFM topographies of Aβ(1-40) after 48 hours of co-incubation with L-NIBC. Panels C and D show the AFM topographies of Aβ(1-40) after 48 hours of co-incubation with gold nanoparticles (L-NIBC-modified) with average particle sizes of 6.0 nm and 3.6 nm, respectively. And panel E shows the AFM topographies of Aβ(1-40) after 48 hours of co-incubation with AuC (L-NIBC-modified) with an average particle size of 1.8 nm.

[0067] In Figure 5, the amyloidosis kinetic curves of Aβ(1-40) at various concentrations of L-NIBC are shown in panel A. The amyloidosis kinetic curves of Aβ(1-40) at various concentrations of gold nanoparticles with sizes of 18.2 nm, 10.1 nm, 6.0 nm, and 3.6 nm are shown in panels B to E. The amyloidosis kinetic curves of Aβ(1-40) at various concentrations of AuC with sizes of 2.6 nm, 1.8 nm, and 1.1 nm are shown in panels F to H. The Aβ amyloidosis kinetic curves in panels A to H are curves when Aβ(1-40) was co-incubated with gold nanoparticles or AuC at various concentrations, where □ represents 0 ppm (i.e., no gold nanoparticles or AuC), ○ represents 0.1 ppm, △ represents 1 ppm, ▽ represents 5 ppm, ◇ represents 10 ppm, and ☆ represents 20 ppm.

[0068] As can be seen from Figure 4, Aβ fibrils were spread throughout panel A as a control, and panel B was the same. Although the fibrils were reduced to some extent, long fibrils were still visible in panel C. Although long fibrils were absent, many short Aβ fibrils were still present in panel D. L-NIBC was shown to have no obvious effect on the formation of Aβ(1-40) fibrils. The addition of small-sized L-NIBC-modified gold nanoparticles could delay but not completely inhibit the Aβ(1-40) amyloidosis process, because the short fibrils continued to grow into long fibrils after further time. The absence of long or short fibrils in panel E of Figure 4 suggests that L-NIBC-modified AuC can completely inhibit the Aβ(1-40) amyloidosis process.

[0069] Figure 4 is a qualitative experiment, while Figure 5 is a quantitative experiment. The results in Figure 5 show that the addition of L-NIBC had no apparent effect on the kinetics of Aβ(1-40) amyloidosis (Panel A of Figure 5), and for gold nanoparticles with particle diameters of 10.1 nm or greater, the addition of L-NIBC significantly reduced the kinetics of Aβ(1-40) amyloidosis. The addition of L-NIBC-modified gold nanoparticles accelerated both the growth and platform phases of Aβ aggregation kinetics (at a gold nanoparticle concentration of 20 ppm, the growth phase of Aβ aggregation kinetics was advanced until the 12th hour, and the platform phase was advanced until the 16th hour), suggesting that L-NIBC-modified gold nanoparticles could accelerate Aβ aggregation (Figure 5, Panels B and C). Furthermore, when gold nanoparticle diameters were 6.0 nm or less (Figure 5, Panels D and E), the onset of Aβ aggregation was delayed (at a gold nanoparticle concentration of 20 ppm, the growth phase of Aβ aggregation kinetics was delayed until the 54th hour), suggesting that gold nanoparticles have an inhibitory effect on Aβ aggregation. However, Figure 5 also shows that the addition of L-NIBC-modified gold nanoparticles, even at a very high concentration (20 ppm), did not completely inhibit Aβ aggregation (i.e., the growth phase did not appear and the fluorescence curve was completely flat). On the other hand, after the addition of L-NIBC-modified gold nanoparticles, the fluorescence emission peak of ThT is located at 515 nm, while the plasmon resonance absorption peak of L-NIBC-modified gold nanoparticles is located near 520 nm. Therefore, the observed decrease in ThT fluorescence intensity is due to partial quenching of the plasmon resonance effect of the gold nanoparticles on ThT fluorescence and should not be due to the inhibitory effect of L-NIBC-modified gold nanoparticles on Aβ(1-40) aggregation.

[0070] Panels F–H of Figure 5 show that all L-NIBC-modified AuCs were able to significantly inhibit Aβ aggregation (the onset of the growth phase was delayed; at a concentration of 5 ppm, the onset of the growth phase in the aggregation kinetics of 20 μM Aβ was delayed by more than 50 h). At concentrations of 10 ppm or higher, Aβ aggregation was completely inhibited (the growth phase did not appear, and the fluorescence curve was completely flat). The minimum concentration of L-NIBC-modified AuC required for complete inhibition is related to the ligand type and AuC diameter. The minimum concentrations of L-NIBC-modified AuC with sizes of 1.1 nm, 1.8 nm, and 2.6 nm were 5.0 ppm, 5.0 ppm, and 10.0 ppm, respectively. Furthermore, because L-NIBC-modified AuCs do not have a plasmon resonance effect, they do not have a quenching effect on ThT fluorescence. Therefore, the decrease in fluorescence intensity observed here was solely due to the inhibitory effect of L-NIBC-modified AuC on Aβ(1-40) aggregation. The quantitative results in Figure 5 are in good agreement with the qualitative results in Figure 4.

[0071] This experiment demonstrated that when the size of L-NIBC-modified gold nanoparticles was 6.0 nm or less, the gold nanoparticles had a certain inhibitory effect on Aβ aggregation and fibrillation, but it was limited. Furthermore, L-NIBC-modified AuC completely inhibited Aβ aggregation and fibrillation. Because the L-NIBC molecule itself cannot affect Aβ aggregation and fibrillation (see Panel B of Figure 4 and Panel A of Figure 5), this function was attributed to AuC, not the L-NIBC ligand. This provides a basis for developing medicines for diseases associated with Aβ aggregation and fibrillation that can be classified as AuC-containing substances as defined by the present invention.

[0072] This embodiment also confirms the functionality of AuC modified with other ligands listed in Table 1. For example, panels A to H of Figure 12 show the inhibitory effects of AuC (at a dose of 10 ppm) modified with CR, N-acetyl-L-cysteine ​​(L-NAC), GSH, 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap), D-NIBC, RC, or L-cysteine ​​and D-cysteine ​​on the aggregation and fibrillation of Aβ(1-40). Similar phenomena were observed for AuC modified with various ligands, leading to the same conclusion. These ligands themselves cannot affect Aβ aggregation and fibrillation, and ligand-modified gold nanoparticles with a size larger than 3 nm only have limited inhibitory effects on Aβ aggregation and fibrillation. Although larger gold nanoparticles may even promote Aβ aggregation and fibrillation, ligand-modified AuC has excellent inhibitory effects against Aβ aggregation and fibrillation, and complete inhibition can be achieved when the concentration exceeds 5 ppm to 10 ppm, although the minimum concentration required for complete inhibition varies slightly depending on the particle size of the ligand and AuC. Similarly, these ligand-modified AuC are classified as AuC-containing substances as defined in the present invention.

[0073] Embodiment 4: Experiments with Aβ-induced AD cell models Cell viability was used as an index in the experiment of this embodiment. The test results of the CCK-8 method reflected the effect of the ligand-modified AuC or gold nanoparticle samples on the toxicity of Aβ(1-40), indicating whether the ligand-modified AuC or gold nanoparticles have a neuroprotective effect on the pathogenesis of amyloid protein misfolding. The cells used in the experiment were SH-SY5Y neuroblastoma cell line. The Aβ-induced AD cell model was established according to the literature (R. Liu, H. Barkhordarian, S. Emadi, CB Park, MR Sierks, Neurobiology of Disease 2005, 20, 74). Specific method: 1) SH-sy5y cells in the logarithmic growth phase (cells subcultured for 6 generations) were diluted with complete medium (MEM + 10% FBS + 1% penicillin-streptomycin) to give 5 x 10 4 A cell suspension was obtained at a density of 100 μL / mL. The suspension was inoculated into a 96-well plate at 200 μL per well and cultured at 37°C in an incubator with 5% CO. When the cells had adhered to the wells, the sample was added.

[0074] 2) 100 μL of ligand-modified AuC samples or ligand-modified gold nanoparticle samples dissolved in maintenance medium (MEM + 2% FBS + 1% penicillin-streptomycin) with various particle sizes and concentrations of 0.04 ppm, 0.4 ppm, 4 ppm, 20 ppm, 40 ppm, and 80 ppm were added to the incubated suspension obtained from step 1. After incubation in an incubator for 2 hours, 100 μL of 80 μM Aβ(1-40) was added, and the mixture was then incubated in an incubator for 24 hours. Thus, the final concentrations of ligand-modified AuC or ligand-modified gold nanoparticles were 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, and 20 ppm, respectively, while the final concentration of Aβ(1-40) was 20 μM. The blank control group contained no SH-sy5y cells; the negative control group contained SH-sy5y cells but no ligand-modified AuC or ligand-modified gold nanoparticles and Aβ(1-40); the cell model control group contained only SH-sy5y cells and Aβ(1-40) (final concentration: 20 μM); and the ligand control group contained SH-sy5y cells, Aβ(1-40) (final concentration: 20 μM), and L-NIBC (final concentration: 20 ppm). The culture medium was removed, and 100 μL of maintenance medium (MEM) containing 10% CCK-8 was added per well. The cells were incubated for 4 hours. The absorbance at 450 nm of each well was measured to reflect the preventive and therapeutic effects of ligand-modified AuC on Aβ(1-40) pathology.

[0075] Taking the L-NIBC-modified AuC in embodiment 2 as an example, L-NIBC-modified gold nanoparticles were compared with AuC, and the results are shown in FIG.

[0076] Panels A to C of Figure 6 show the effects of L-NIBC-modified AuC with particle sizes of 1.1 nm, 1.8 nm, or 2.6 nm, respectively, on cell viability in an Aβ-induced AD cell model at various concentrations, and panels D to F show the effects of L-NIBC-modified gold nanoparticles with particle sizes of 3.6 nm, 6.0 nm, or 10.1 nm, respectively, on cell viability in an Aβ-induced AD cell model at various concentrations.

[0077] As shown in Figure 6, the addition of L-NIBC alone did not improve cell viability. L-NIBC-modified AuCs with various sizes (average sizes were 1.1 nm, 1.8 nm, and 2.6 nm, respectively) increased cell viability from approximately 60% to over 95% in the Aβ-induced AD cell model, even at very low doses (e.g., 0.1 ppm to 1 ppm) (P values ​​were all less than 0.05 in Panels A to C of Figure 6). L-NIBC-modified gold nanoparticles with an average diameter of 3.6 nm increased cell viability to some extent with increasing concentrations in the AD cell model (Panel D of Figure 6), but not significantly (P > 0.05). L-NIBC-modified gold nanoparticles with average diameters of 6.0 nm and 10.1 nm, respectively, had no effect on cell viability (Panels E and F of Figure 6). The above results indicated that L-NIBC-modified AuC had significant efficacy in the Aβ-induced AD cell model, whereas L-NIBC-modified gold nanoparticles had no obvious efficacy.

[0078] Experiments with AuCs modified with other ligands listed in Table 1 at various sizes were also carried out in this embodiment. The results also showed that the ligand-modified AuCs significantly improved cell viability in an Aβ-induced AD cell model. It was shown that AuCs modified with various ligands have excellent therapeutic effects on AD, at least at the cell model level, and can be classified as AuC-containing substances as defined in the present invention and used for AD treatment.

[0079] Embodiment 5: Experiments with AD transgenic mouse models Test 1: 1) 1.0 g of each AuC modified with the ligands listed in Table 1 was weighed out and dissolved in 100 mL of water as a stock solution, which was then stored at 4°C for later use. A small volume of the stock solution was taken and diluted in water before use.

[0080] 2) 180 transgenic mice of the B6 / J-Tg(APPswe,PSEN1de9)85Dbo / MmNju strain (purchased from the Model Animal Research Center of Nanjing University) were randomly divided into three groups, including a control group, a low-dose group, and a high-dose group, with 60 mice per group. When the mice were 100 days old, the mice in the control group were fed normally daily, the mice in the low-dose group were orally administered 200 μL of 0.5 g / L AuC in water per day, and the mice in the high-dose group were orally administered 200 μL of 2 g / L AuC in water per day.

[0081] 3) The mice in the control group, low-dose group, and high-dose group were randomly divided into seven groups. When the mice reached 140, 160, 180, 200, 230, 260, and 290 days of age, maze experiments, open-field experiments, and novel object recognition experiments were conducted to study changes in the mice's learning and memory behaviors. In the first four groups, each group contained six mice, and in the last three groups, each group contained six to eight mice (considering the mortality rate during the feeding process).

[0082] 4) After behavioral studies of the mice in each group, the Aβ content in the blood was detected. Blood was collected from the orbital venous plexus, and the Aβ content and Aβ aggregation were detected by serum ELISA.

[0083] 5) After detecting the Aβ content in the blood of each group of mice, Aβ amyloid deposits distributed in the hippocampus were detected. The mice were anesthetized after obtaining ocular blood and fixed via cardiac perfusion. The whole brains of the mice were collected and sedimented in a sucrose gradient. The brains were then analyzed. The distribution of Aβ amyloid deposits within the hippocampus was examined by immunohistochemistry.

[0084] These results indicated that the ligand-modified AuC provided in the present invention can significantly improve the cognitive behavior of AD transgenic mice and suppress the formation of senile plaques in the brain and the occurrence of the disease, and therefore it can be used as an AuC-containing substance to treat AD.

[0085] Test 2: 1. 1.0 g of each AuC modified with the ligands listed in Table 1 was weighed and dissolved in 100 mL of water as a stock solution, which was then stored at 4°C for future use. A small volume of the stock solution was taken and diluted in water before use. The stock solution was prepared once every two weeks.

[0086] 2. Ninety transgenic mice of the B6 / J-Tg(APPswe,PSEN1de9)85Dbo / MmNju strain (purchased from the Model Animal Research Center of Nanjing University) were randomly divided into three groups, each with 30 mice: a model control group, a low-dose group, and a high-dose group. (Considering that this strain of transgenic mice has a mortality rate of approximately 30% during the feeding process, more mice were present initially than at the end of the experiment to ensure sufficient mice at the end of the experiment.) When the mice reached 100 days of age, the mice in the model control group were fed normally daily, while the mice in the low-dose and high-dose groups were orally administered AuC solution at doses of 5 mg / kg body weight and 20 mg / kg body weight via intraperitoneal injection once every two days, respectively.

[0087] 3. The cognitive behavior of mice was tested using a water maze experiment. The Morris water maze (MWM) experiment involves swimming animals to learn to find a hidden platform in water. The MWM is primarily used to test the learning and memory abilities of animals in spatial location and direction perception, and is widely used in the development and evaluation of AD drugs. A shorter escape latency, a higher number of crossings after the platform is removed, a longer swimming distance in the target quadrant, and a longer time spent in the target quadrant indicate that the mouse has better memory abilities in spatial location and direction perception. 150 days after administration, the behavior of each mouse was tested in a Morris water maze experiment. The experimental method was described in the literature (CV Vorhees, MT Williams, Nature Protocols 2006, 1, 848). The details were as follows:

[0088] (1) Positioning navigation experiment: MWM The test system consisted of a circular pool and an automated video and analysis system. A camera above the pool was connected to a computer (see Figure 13). The water maze consisted of a circular pool with a diameter of 120 cm and a height of 60 cm, and a platform with a diameter of 9 cm. The liquid level was 0.5 cm higher than the platform, and the water temperature was 22 ± 0.5°C. White dye was used to dye the water opalescent. The location experiment was used to measure the learning and memory abilities of mice in the water maze, which continued for 4 days. As shown in Figure 13, the water maze was divided into four quadrants in the shape of a cross, with four directions: east (E), west (W), south (S), and north (N). The platform was located in the center of the SW quadrant, and its position was fixed throughout the experiment. During training, mice were gently placed in water from 1 / 2 radian in different quadrants, with their heads facing the pool wall and close to the inner wall. The time the mouse spent climbing onto the hidden platform (escape latency) was recorded by a camera tracking system or the experiment was stopped when the recording time reached 60 seconds. After climbing onto the platform, the mouse remained on the platform for 30 seconds. If the mouse failed to find the platform within 60 seconds (the escape latency was recorded as 60 seconds), the experimenter guided the mouse to climb onto the platform and allowed it to remain there for 30 seconds. After the experiment, all mice were removed and gently patted dry. Each mouse was trained four times per day for four consecutive days, with 15-20 minutes between training sessions.

[0089] (2) Spatial exploration test: After completing the training on the fourth day, the platform was removed on the fifth day, and the mouse was gently placed in the water from the midpoint of the NE arc (the point farthest from the platform) facing the pool wall. The movement trajectory of the mouse for 60 seconds was recorded by a camera, and the number of times the mouse crossed the platform, the time it stayed in the target quadrant, and the swimming distance in the target quadrant were analyzed by software.

[0090] 4. Immunohistochemistry was used to detect the distribution of Aβ(1-40) and Aβ(1-42) amyloid deposits in the hippocampus and cerebral cortex. Pathological deposition of Aβ outside neurons in the cerebral cortex and hippocampus is a major pathological feature of AD. In particular, Aβ(1-40) and Aβ(1-42) are important components of senile plaques in the brain, which are neurotoxic and can cause progressive cognitive dysfunction and memory loss. In this experiment, changes in Aβ(1-40) and Aβ(1-42) plaque formation in the hippocampus and cerebral cortex were investigated using immunohistochemistry.

[0091] Specific Methods: After continuous administration of the drug for 100 and 150 days, 10 to 12 mice were selected from each group and subjected to immunohistochemistry of the hippocampus and cerebral cortex. The mice administered for 150 days were those that had completed the MWM experiment. The mice were anesthetized with an intraperitoneal injection of 5% chloral hydrate (10 μL / g), their limbs were immobilized on the laboratory table, and the chest was opened to fully expose the heart. Care was taken not to cut the liver during the thoracotomy. The left ventricle was first washed with 50 mL of 0.1 mol / L PBS for 5 minutes to remove blood, and then perfusion-fixed with 4% paraformaldehyde in 0.1 mol / L PBS for 6 minutes. After perfusion fixation, the brains were removed and placed in 4% paraformaldehyde at 4°C for overnight fixation. The tissues were dehydrated using a gradient of 10%, 20%, and 30% sucrose solutions and stored at -80°C for later use. The tissues were embedded in paraffin. The midbrain, hippocampus, and cerebral cortex (8 μm thick) were sliced ​​according to the mouse brain map and used for immunohistochemical staining. The process was as follows: Frozen 8 μm-thick slices were kept at room temperature for 30 minutes, fixed in acetone at 4°C for 20 minutes, washed three times with PBS (5 minutes each time), and then incubated in 3% H2O2 for 10 minutes to eliminate peroxidase activity. After washing three times with PBS (5 minutes each time), the slices were blocked with 10% normal goat serum at room temperature for 40 minutes (slices used for Aβ(1-42) immunohistochemistry were incubated in 10% formic acid for 10 minutes before blocking to restore antigen activity). The serum was discarded, and anti-Aβ(1-40) (ab20068, 1:20 dilution) or anti-Aβ(1-42) working solution (ab12267, 1:200 dilution) was added to the slices. They were incubated at room temperature for 2 hours and washed three times with PBS (5 minutes each time). Horseradish enzyme-labeled streptavidin (diluted in PBS) was added dropwise to the secondary antibody working solution and incubated at room temperature for 1 hour.After washing with PBS three times (5 minutes each time), a nickel sulfate-sensitized DAB blue reaction method was used for color development for 10 minutes. When the positive product was dark blue and the background was clear, it was rinsed three times with distilled water to stop the color development. After counterstaining with hematoxylin for 1 minute, it was rinsed with tap water, dried in a ventilated place, and sealed with neutral gum. The number of Aβ plaques in the whole hippocampus and cerebral cortex was observed under a confocal microscope. The number of ventricles was counted by mirror. Each sample contained two slices containing both the left and right ventricles as parallel samples. The average value was calculated for statistical analysis. All data were processed using SPSS software (SPSS 21), and t-tests or one-way analysis of variance were performed. P<0.05 indicated a statistically significant difference.

[0092] Taking the L-NIBC-modified AuC with an average size of 1.8 nm in embodiment 2 as an example, the results of the water maze experiment 150 days after administration are shown in Figure 14. The results showed that there was no statistical difference in escape latency between the mice in the model control group and the high-dose and low-dose groups on days 1 and 2 of the place-navigation test (P>0.05, n=10-12 mice / group) (Panel A of Figure 14). With increasing training time, the escape latency of the mice in the high-dose group was significantly shorter than that of the model group on days 3 and 4 (P<0.01 and P<0.05), and the escape latency of the low-dose group was shorter than that of the model group, but there was no statistical difference (P>0.05, see Panel A of Figure 14). After the place-navigation experiment of the mice was completed, the platform was removed and a space search experiment was conducted. The results showed that, compared with the mice in the model control group, the mice in the high-dose group showed a significant increase in the number of platform crossings and swimming distance in the target quadrant (P<0.05), and also a significant increase in the time spent in the target quadrant (P=0.05). Compared with the mice in the model control group, the mice in the low-dose group showed an increase in the number of platform crossings, swimming distance in the target quadrant, and time spent in the target quadrant, but the differences were not significant (P>0.05) (Figure 14, Panels B to D). The above results showed that after 150 days of AuC administration, AuC significantly improved the ability of APP / PS1 mice to learn and remember spatial location and direction perception. This effect was dose-dependent.

[0093] The results of immunohistochemical experiments to detect the distribution of amyloid deposits of Aβ(1-40) and Aβ(1-42) in the hippocampus and cerebral cortex are shown in FIGS.

[0094] Panels A, B, and C of Figure 15 show representative immunohistochemical slice results for Aβ(1-40) in the hippocampus and cerebral cortex in the high-dose, low-dose, and model control groups on day 100 of administration. Panel D of Figure 15 shows statistical results. The experimental results showed that, compared with the model control group, mice in the high-dose group had a significant reduction in Aβ(1-40) plaques in the hippocampus (44.6 ± 12.2%, P < 0.05) but not in the cerebral cortex (P > 0.05) on day 100 of administration. The low-dose group had no significant effect on the formation of Aβ(1-40) plaques in the hippocampus and cerebral cortex (P > 0.05). Figure 16 shows the corresponding results for Aβ(1-42). These results showed that high-dose administration significantly reduced Aβ(1-42) plaque formation in the cerebral cortex (by 61.5±11.4%, P<0.05), but not in the hippocampus (P>0.05). Low-dose administration had no significant effect on Aβ(1-42) plaque formation in the hippocampus or cerebral cortex (P>0.05). These results indicated that on the 100th day of administration, AuC had a significant inhibitory effect on Aβ(1-40) and Aβ(1-42) plaque formation, and this effect showed a clear dose-dependent relationship.

[0095] With increasing administration time and mouse age, the formation of Aβ(1-40) and Aβ(1-42) plaques in the hippocampus and cerebral cortex of mice significantly increased in the model control group at 150 days of administration compared with 100 days of administration. Specifically, Aβ(1-40) increased by 57.2±7.2% in the hippocampus (P<0.05) and 49.1±19.6% in the cerebral cortex (P<0.05), and Aβ(1-42) increased by 74.4±7% in the hippocampus (P<0.05). In the rat model, the Aβ(1-40) levels increased by 65±11.1% (P<0.05) in the hippocampus and cerebral cortex, respectively, after 150 days of treatment. The results suggest that the older the model mice, the greater the impact on memory and cognitive function. Panels A, B, and C of Figure 17 show representative immunohistochemical slice results for Aβ(1-40) in the hippocampus and cerebral cortex in the high-dose group, low-dose group, and model control group, respectively, on day 150 of treatment. Panel D of Figure 17 shows the statistical results. The results showed that in the high-dose group, Aβ(1-40) was obviously reduced in both the hippocampus and cerebral cortex of mice (reduced by 59.0±11.1% (P<0.05) in the hippocampus and by 36.4±4.5% (P<0.05) in the cerebral cortex), while administration at a low dose had no significant effect on the formation of Aβ(1-40) plaques in the hippocampus (P>0.05), but significantly reduced Aβ(1-40) plaques in the cerebral cortex (26.9±2.1% (P<0.05)). AuC was shown to have a significant inhibitory effect on the formation of Aβ(1-40) plaques on day 150. This effect was also observed after administration. A dose-dependent relationship was observed. Furthermore, using SPSS software, we analyzed the correlation between the number of Aβ(1-40) plaques and the number of attempts to cross the platform in the water maze experiment on day 150. The analysis revealed that the number of Aβ(1-40) plaques in the hippocampus and cerebral cortex had a significant negative correlation with the number of attempts to cross the platform (hippocampus: R = -0.848, P < 0.01; cerebral cortex: R = -0.802, P < 0.05). Furthermore, these results confirmed the correlation between the AuC-induced reduction of Aβ(1-40) plaques in the hippocampus and cerebral cortex and the AuC-induced improvement in memory and learning abilities in mice.

[0096] Figure 18 shows the corresponding results of Aβ(1-42) after 150 days of AuC administration. The results showed that high-dose AuC administration significantly inhibited Aβ(1-42) plaque formation in the hippocampus and cerebral cortex (reduced by 51.1 ± 6.7% (P < 0.05) in the hippocampus and 62.8 ± 4.6% (P < 0.05) in the cerebral cortex). Low-dose administration had no significant effect on Aβ(1-42) plaque formation in the hippocampus and cerebral cortex of mice (P > 0.05). AuC was shown to have a significant inhibitory effect on Aβ(1-42) plaque formation at 150 days. This effect showed a dose-dependent relationship. Correlation statistical analysis using SPSS revealed that the number of Aβ(1-42) plaques in the hippocampus and cerebral cortex was significantly negatively correlated with the number of platform crossings (hippocampus: R = -0.794, P < 0.05, cerebral cortex: R = -0.802, P < 0.05). Furthermore, the results confirmed the correlation between the AuC-induced reduction of Aβ(1-42) plaques in the hippocampus and cerebral cortex and the AuC-induced improvement in memory and learning abilities in mice.

[0097] In summary, AuC could significantly improve the cognitive behavior of AD model mice and inhibit the formation of Aβ(1-40) and Aβ(1-42) plaques in the hippocampus and cerebral cortex. Therefore, AuC could suppress the onset of pathology in diseased mice and could be used as an AuC-containing substance for the prevention and treatment of AD.

[0098] AuC modified with other ligands listed in Table 1 have similar effects, so they were not described in detail here.

[0099] Embodiment 6: Experiments on the aggregation kinetics of α-syn in vitro This embodiment confirms the functionality of the ligand-modified AuC through in vitro α-syn aggregation kinetics experiments and compares it to the effect of the ligand molecule alone on the aggregation kinetics of α-syn, thus demonstrating that the functionality is due to AuC and not the ligand.

[0100] Thioflavin T (ThT) is a dye that specifically stains amyloid fibrils. When ThT is incubated with a polypeptide or protein monomer, its fluorescence does not change significantly. When ThT encounters an amyloid polypeptide or protein with a fibrillar structure, it immediately binds to the amyloid polypeptide or protein, resulting in an exponential increase in its fluorescence intensity. For this reason, ThT is widely used as a marker for monitoring peptide or protein amyloidosis. This embodiment utilizes the ThT fluorescent labeling method to monitor the dynamic process of α-syn fibrillary aggregation in the presence of AuC. The specific experimental method was as follows.

[0101] Pretreatment of α-syn monomer: Lyophilized α-syn powder (Bachem Corp.) was dissolved in HFIP to obtain a 1 g / L α-syn solution. The solution was sealed and incubated at room temperature for 2 to 4 hours. The HFIP was then blown dry with high-purity nitrogen in a fume hood. Finally, the dried α-syn was dissolved in 200 μL of DMSO. After sealing, the solution was kept at −20°C in a refrigerator for up to 1 week for further use. Before use, the α-syn DMSO solution was diluted with a large amount of phosphate buffer solution (PBS, 10 mM, pH = 7.4) until the α-syn concentration reached 20 μM, thereby obtaining a PBS solution of α-syn. All α-syn PBS solutions in this experiment were freshly prepared.

[0102] Sample preparation and detection: Ligand-modified AuC at various concentrations listed in Table 1 was added to a 35 μM α-syn solution in PBS and continuously incubated in a 96-well plate at 37 °C using ThT fluorescent labeling. The fluorescence intensity was monitored every 10 minutes using a microplate reader. The kinetics of α-syn aggregation was characterized by changes in ThT fluorescence intensity. For example, L-NIBC-modified AuC with a particle size of 1.8 nm prepared in Example 2 was used as the experimental group. L-NIBC molecules not bound to AuC were used as the ligand control group. Four concentrations of AuC were employed: 0 ppm (a model control group containing only α-syn, without AuC or L-NIBC), 1.0 ppm, 5.0 ppm, and 10.0 ppm. L-NIBC molecules were used at two concentrations: 1.0 ppm and 10.0 ppm.

[0103] The results are shown in Figure 19. The results showed that during incubation of 35 μM α-syn at 37°C, the fluorescence intensity of ThT-labeled α-syn rapidly increased from 48 hours later. This confirmed the occurrence of α-syn aggregation and fibrillization. This was consistent with the results reported in the literature (VN Uversky, J. Li, P. Souillac, I.S. Millett, S. Doniach, R. Jakes, M. Geodert, A.L. Fink, Journal of Biological Chemistry 2002, 277, 11970). The results of the ligand control group showed that the use of L-NIBC alone had no obvious effect on the kinetics of α-syn aggregation (Panel A of Figure 19). In the experimental groups to which AuC was added at low concentrations (e.g., 1.0 ppm and 5.0 ppm), the ThT-labeled fluorescence intensity was significantly reduced and the onset time was significantly delayed compared to the model control group and the ligand control group (Figure 19, Panel B). This suggests that the addition of AuC can significantly inhibit α-syn aggregation and fibrillization. When the AuC concentration reached 10 ppm, the ThT-labeled fluorescence intensity remained near the baseline without any increase throughout the 168-hour experiment (Figure 19, Panel B). This suggests that α-syn aggregation and fibrillization can be completely inhibited when the AuC concentration is sufficiently high.

[0104] In this experiment, AuC modified with various other ligands listed in Table 1 were also investigated. For example, in panels C to J of Figure 19, D-NIBC, CR, RC, 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap) The inhibitory effects of AuC modified with GSH, N-acetyl-L-cysteine ​​(L-NAC), L-cysteine ​​(L-Cys), and D-cysteine ​​(D-Cys) (all at 10 ppm) on α-syn aggregation and fibrillation were demonstrated. Similar phenomena were observed for AuC modified with various ligands, and the same conclusions were reached. Although these ligands themselves may not affect α-syn aggregation and fibrillation, the ligand-modified AuC had excellent inhibitory effects on α-syn aggregation and fibrillation. When the concentration reached 10 ppm, all of them achieved complete inhibition. The minimum concentration required for complete inhibition varied slightly for various ligands. Similarly, these ligand-modified AuC were classified as AuC-containing substances as defined in this invention. Other AuCs listed in Table 1 had similar effects. Only the AuC concentration required for complete inhibition of α-syn aggregation and fibrillation differed. It will not be described in detail.

[0105] Embodiment 7: MPP + Experiments with the induced PD cell (SH-sy5y) model Test 1: This experiment uses cell viability as an indicator. The results obtained from the CCK-8 assay showed that ligand-modified AuC or gold nanoparticles significantly inhibited MPP in the SH-sy5y neuronal model of PD. + This reflects their resistance to the toxic effects of MPP (commonly used neurotoxins), thus supporting their neuroprotective effect in PD. + The induced PD cell model has been described in the literature (Cassarino, DS; Fall, CP; Swerdlow, RH; Smith, TS; Halvorsen, E The method was established as described in [M; Miller, SW; Parks, JP; Parker, WD Jr; Bennett, JP Jr. Elevated reactive oxygen species and antioxidant enzyme activities in animal and cellular models of Parkinson's disease. Biochimica et biophysica acta. 1997.1362.77-86]. The specific method was as follows.

[0106] 1) SH-sy5y cells in logarithmic growth phase were diluted with complete medium to 5 x 10 4 A cell suspension was obtained at a cell density of 100 μL / mL. The suspension was inoculated into a 96-well plate at 200 μL per well and cultured at 37°C in an incubator with 5% CO. When the cells had adhered to the wells, the sample was added.

[0107] 2) 100 μL of ligand-modified AuC samples (listed in Table 1) or ligand-modified gold nanoparticle samples with various particle sizes and concentrations were dissolved in the maintenance medium and added as the first group to final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, and 20 ppm, respectively. The first group was the treatment group. Two hours after pretreatment with ligand-modified AuC or gold nanoparticles, MPP + (final concentration was 1 mM) was added simultaneously to the treatment group and the cell control group, respectively. The blank control group was a group that did not contain SH-sy5y cells, and the negative control group contained SH-sy5y cells but did not contain AuC or gold nanoparticles and MPP. + The cell control group was a group containing SH-sy5y cells and 1 mM MPP. + The ligand control group contained SH-sy5y cells and 1 mM MPP. +and a group containing the corresponding ligand molecule (final concentration was 20 ppm). Then, the samples in all groups were incubated at 37°C for 24 hours, centrifuged to remove the culture medium, 100 μL of maintenance medium containing 10% CCK-8 was added to each well, and the incubation was continued for 4 hours. After that, the absorbance of each well was measured at 450 nm to determine the MPP of the ligand-modified AuC. + This reflected a pre-protective effect and a healing effect on the lesions.

[0108] The same procedure was adopted to carry out experiments for AuC and gold nanoparticles modified with various ligands. The results showed that the ligand-modified AuC provided in the present invention has a neuroprotective effect against PD. This effect was also due to the ligand-modified AuC. These results suggest that AuC-containing materials can be used to suppress PD.

[0109] Test 2: This experiment uses cell viability as an indicator. The results obtained from the CCK-8 assay showed that ligand-modified AuC or gold nanoparticles significantly inhibited MPP in the SH-sy5y neuronal model of PD. + This reflects the resistance to the toxic effects of MPP (commonly used neurotoxins) and supports their neuroprotective effect in PD. + The induced PD cell model is established as described in the reference (DS Cassarino, CP Fall, RH Swerdlow, TS Smith, EM Halvorsen, SW Miller, JP Parks, WD Jr. Parker, JP Jr. Bennett, Biochimica et Biophysica Acta 1997, 1362, 77). Specific method: 1) SH-sy5y cells in logarithmic growth phase were diluted with complete medium to 5 x 10 4A cell suspension was obtained at a cell density of 100 μL / mL. The suspension was inoculated into a 96-well plate at 200 μL per well and cultured at 37°C in an incubator with 5% CO. When the cells had adhered to the wells, the sample was added.

[0110] 2) 100 μL of ligand-modified AuC samples (listed in Table 1) or ligand-modified gold nanoparticle samples with various particle sizes and concentrations were dissolved in the maintenance medium and added as the first group to final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, and 20 ppm, respectively. The first group was the treatment group. Two hours after pretreatment with ligand-modified AuC or gold nanoparticles, MPP + (final concentration was 1 mM) was added simultaneously to the treatment group and the cell control group, respectively. The blank control group was a group that did not contain SH-sy5y cells, and the negative control group contained SH-sy5y cells but did not contain AuC or gold nanoparticles and MPP. + The cell control group was a group containing SH-sy5y cells and 1 mM MPP. + The AuC control group and the ligand control group were SH-sy5y cells and 1 mM MPP. + and a group containing the corresponding ligand molecule (final concentration was 20 ppm). Then, the samples in all groups were incubated at 37°C for 24 hours, centrifuged to remove the culture medium, 100 μL of maintenance medium containing 10% CCK-8 was added to each well, and the incubation was continued for 4 hours. After that, the absorbance of each well was measured at 450 nm to determine the MPP of the ligand-modified AuC. + This reflected a pre-protective effect and a healing effect on the lesions.

[0111] The experimental results of L-NIBC modified AuC or gold nanoparticles were interpreted as shown in Figure 20. These results show that after 24 hours of culture, MPP +The cell viability of the AuC control group, to which 100 mM AuC was added without MPP, was shown to be increased to 108.5 ± 7.1% (P < 0.01) compared to the blank control group (defined as 100%), suggesting that AuC is non-toxic. + The cell viability of the model control group, which was treated with MPP but without AuC, decreased to 65.1±4.0% (P<0.01 vs. the blank control group), while the cell viability of the ligand control group was 61.5±3.8% (P<0.01 vs. the blank control group). Therefore, the ligand alone was effective in preventing the cell viability of MPP + This suggests that the ligand-modified AuC provided in the present invention has a protective effect on neurons in PD, and this effect is also due to AuC rather than the ligand. On the other hand, the corresponding gold nanoparticles with the same ligand did not significantly improve the viability of the model cells at all experimental concentrations. The cell viability of the treatment groups with 1 ppm, 5 ppm, 10 ppm, and 40 ppm AuC was increased to 97.9±2.8% (vs. model control group, P<0.01), 99.7±4.0% (vs. model control group, P<0.001), 95.3±1.7% (vs. model control group, P<0.01), and 93.2±0.4% (vs. model control group, P<0.01), respectively. Therefore, gold nanoparticles cannot be used as a medicine for the prevention and treatment of PD.

[0112] The same procedure was adopted to carry out experiments for AuC modified with various ligands listed in Table 1. Their effects were similar, so they will not be described in detail here.

[0113] Embodiment 8: MPP + Experiments with induced PD cell (PC12) model MPP in PC12 cells +A model of apoptosis induced by AuC (100 mM) was used in combination with flow cytometry techniques to determine MPP of AuC in this experiment. + The protective effect against induced cell injury and apoptosis was observed. Specific methods: The blank control group was MPP + and a group without AuC added, MPP + The model group is MPP + The AuC control group was a group to which only AuC was added, and the experimental group was a group to which only MPP + In the experimental group, a solution of L-NIBC-modified AuC with an average particle size of 1.8 nm was added to the PC12 cell suspension (the final concentration of AuC was 20 ppm), and after half an hour, MPP + The mixture was incubated for 24 hours and analyzed by Annexin V-FITC / PI Cell Apoptosis Detection Kit (purchased from Roche) and FACSCalibur flow cytometry. A cytometer was used to detect cell proliferation activity and apoptosis, and the data were acquired and analyzed by CellQuest Pro.

[0114] The experimental results are shown in Figure 21. The cell cytometry detection results showed that MPP + After incubation with MPP for 24 hours, + The percentage of cell apoptosis in the blank control group without MPP was 23.5% ± 2.8%. When 20 ppm AuC alone was co-incubated with PC12 cells, the percentage of cell apoptosis was 28.47 ± 3.2%, which showed no significant difference from the blank control group, suggesting that AuC has no obvious cytotoxic effect. + The percentage of cell apoptosis in the model group was 49.5±10.1%, which was significantly increased compared with the blank control group (P<0.001). +The percentage of cell apoptosis was significantly higher when the MPP was added to the 1000 cells for 1 / 2 hour and then co-incubated for 24 hours. + Compared with the model group, the cell apoptosis was significantly reduced to 35.9±2.2% (P<0.05).

[0115] The same procedure was adopted to carry out experiments for AuC modified with various ligands listed in Table 1. Their effects were similar, so they will not be described in detail here.

[0116] The combined results of embodiment 7 and embodiment 8 demonstrate that AuC effectively improves cell viability and promotes MPP + It was shown that it could significantly inhibit cell apoptosis in an induced PD cell model.

[0117] Embodiment 9: Experiments with MPTP-induced PD mouse model Test 1: Experimental animals: 80 C57bl / 6 male mice, 8 weeks old, weighing 25-30g; three mice per cage were housed at room temperature of 22-27°C, with a 12-hour circadian rhythm, and allowed to eat and drink ad libitum. They were allowed to acclimate for 7 days.

[0118] MPTP-induced PD mouse model: Mice were divided into four groups, including a blank control group, an AuC normal control group, an MPTP model group, and an AuC treatment group, with 2 mice per group. The mice were randomly assigned to each group. The MPTP model group and AuC-treated group received four subcutaneous injections of 20 mg / kg (free base) MPTP, one every 2 hours. The blank control group received four subcutaneous injections of 20 mg / kg saline, one every 2 hours. Eight hours after the last injection, the blank control group and MPTP model group received daily intravenous injections of 10 μL saline, while the AuC normal control group and AuC-treated group received daily intraperitoneal injections of 10 μL saline solutions of the ligand-modified AuC listed in Table 1 (AuC concentration: 10 g / L). The injections were continued for 7 days. The animals were placed in a feeding box with clean padding and provided with water and food ad libitum.

[0119] Behavioral Test: Rotarod Test. The rotarod test requires animals to balance and move on a roller. It is a widely used test to test motor coordination. The diameter of the roller is 6 cm, and the rotation speed is 20 rpm. After the animals were adapted to the roller five times, the test was started at 1-minute intervals. The latency to fall from the rotarod was recorded five times consecutively, and the average value was calculated.

[0120] Neurotransmitter Measurement: After behavioral testing, the animals were sacrificed, and the mouse striatum was removed and stored at -80°C. For measurement, the striatum was treated with 10 μL / mg (striatum) of homogenate (0.1 M perchloric acid, 0.1 mM EDTA-2Na), disrupted by ultrasound in an ice bath for 30 minutes, and centrifuged at 10,000 rpm in a refrigerated centrifuge for 10 minutes. The supernatant was removed, filtered through a 0.25 μm filter, and injected into an HPLC liquid chromatography column. The levels of dopamine (DA) transmitter and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum were detected in the laboratory using a high-performance liquid phase system. The chromatography column must be maintained with freshly prepared mobile phase for 2 hours before each test. HPLC conditions: flow rate: 1 mL / min, column temperature: 30°C, excitation wavelength and absorption wavelength of the fluorescence detector were 280 nm and 330 nm, respectively.

[0121] Measurement of tyrosine hydroxylase: Brain tissues were removed and fixed in 4% PFA + 2% sucrose for 4-6 hours, then immersed in 30% sucrose solution. After the tissues sank to the bottom, they were embedded in OCT and sliced ​​into serial coronal sections using a frozen slicer. They were stained with ABC (avidin-biotin-peroxidase complex) method. Frozen tissues of the substantia nigra were removed, sliced, stained in TH, developed with diphenylamine, and observed under a microscope and photographed.

[0122] These results showed that the ligand-modified AuC provided in the present invention could significantly improve the motor behavior of MPTP-induced PD model mice, increase the number of dopaminergic neurons, and improve the brain level of dopamine neurotransmitter, which could be used as an AuC-containing substance to treat PD.

[0123] Test 2: Experimental animals: 80 C57bl / 6 male mice, 8 weeks old, weighing 25-30g; three mice per cage were housed at room temperature of 22-27°C, with a 12-hour circadian rhythm, and allowed to eat and drink ad libitum. They were allowed to acclimate for 7 days.

[0124] MPTP-induced PD mouse model: Mice were randomly divided into four groups, with 20 mice per group, including a blank control group, an AuC control group (classified into a low-dose group and a high-dose group based on the dose of AuC), an MPTP model group, and an AuC treatment group (classified into a low-dose group and a high-dose group based on the dose of AuC). In the MPTP model group and the AuC experimental group, 30 mg / kg (free base) MPTP was administered daily. The animals were intraperitoneally injected once daily for 7 days. The blank control group received subcutaneous injections of 30 mg / kg saline once daily for 7 days. The low-dose AuC control group and the low-dose AuC treatment group received intraperitoneal injections of 100 μL of 1 g / L L-NIBC-modified AuC with an average particle size of 1.8 nm in saline once daily. The high-dose AuC control group and the high-dose AuC treatment group received intraperitoneal injections of 100 μL of 4 g / L L-NIBC-modified AuC with an average particle size of 1.8 nm in saline once daily for 7 days. The animals were placed in a feeding box with clean padding and provided with water and food ad libitum.

[0125] 1. Behavioral Testing: (1) Spontaneous movement test: The animals were transferred from their cages to a locomotor activity detection device. After the animals were allowed to adapt to the new environment for 5 minutes, their spontaneous movement and changes were recorded within 5 minutes. The movement distance and movement speed of the animals within 5 minutes were used to measure the amount of movement of the animals.

[0126] (2) Swimming test: Donnan's test (GA Donnan, GL Willis, SJ Kaczmarezyk, P. Rowe, Journal of the Neurological Science 1987, 77, 185) The mice were placed in a Morris tank with a water depth of 60 cm and a temperature of 22° C. The swimming distance and swimming time of the animals were recorded within 10 minutes to measure their activity.

[0127] (3) Rotarod test: The rotarod test requires animals to balance and move on a roller. This test is widely used to test motor coordination. The diameter of the roller is 6 cm, and the rotation speed is 20 rpm. After the animals were adapted to the roller five times, all tests were performed at 1-minute intervals. The latency to fall from the rotarod was recorded. The tests were performed five times consecutively, and the average value was calculated.

[0128] 2. Immunohistochemical detection in the striatum and substantia nigra: After behavioral testing, five mice from each group were removed and immunohistochemical detection was performed in the substantia nigra and striatum. After abdominal anesthesia with 1 mL of 0.5% sodium pentobarbital, the thorax was opened. Blood was first rinsed from the aorta with 15 mL of 0.9% saline, and then perfused (fast at first, then slow) with 100 mL of 0.1 mol / L phosphate buffer solution (PBS, pH 7.2) containing 4% paraformaldehyde for 1 hour. After perfusion fixation, the brains were removed, placed in 4% paraformaldehyde, embedded in paraffin, and sectioned coronally in the substantia nigra and striatum according to the mouse brain map. Brain slices were 3 μm thick per section. The resulting brain slices were used in ultrasensitive two-step immunohistochemistry by immunofluorescence and other experiments. The immunochemical staining procedure was as follows. The brain slices were placed in a 0.3% HO solution in methanol (1 mL 30% HO + 80 mL methanol + 19 mL PBS) for 30 min, then in 0.3% Triton X-100 in PBS for 30 min. They were then immersed in mouse anti-tyrosine hydroxylase (TH) monoclonal antibody (1:200) or IBa1 (1:250 dilution) for 48 h at 4°C, and then in biotinylated rabbit anti-mouse secondary antibody (1:500) for 2 h at room temperature. They were quickly rinsed with distilled water and developed using the nickel ammonium sulfate-enhanced DAB blue reaction method for 20–30 min. When the positive product was dark blue and the background was clear, the brain slices were rinsed three times with distilled water to stop the color development. After each of the above steps, the brain slices were washed three times with 0.01 mol / L PBS for 10 minutes each time. The primary antibodies used here were diluted in PBS containing 1% bovine serum and 0.3% Triton X-100, and the secondary antibodies and ABC complexes were diluted in PBS. Brain slices were then attached, dehydrated, and mounted with clear neutral gum.

[0129] 3. Protein immunoblotting (WB) assay: Tyrosine hydroxylase (TH) is a key enzyme in the dopamine (DA) biosynthesis pathway. TH immunohistochemistry shows changes in DAergic neurons in the substantia nigra and striatum (D. Luo, J. Zhao, Y. Cheng, S.M. Lee, J. Rong, Molecular Neurobiology 2017, DOI: 10.1007 / s12035-017-0486-6). After behavioral testing, five mice from each group were removed for striatal WB detection. The required brain tissues were taken on ice, crushed in RIPA lysate, and homogenized by centrifugation at 12,000 g for 30 min at 4°C. Proteins were extracted and prepared for SDS-polyacrylamide gel electrophoresis at 55–60 V for 4.5 h. Proteins were then transferred to membranes using the semi-dry method at a constant current of 60 mA for approximately 1.5 h. The membrane was blocked with 5% skim milk at room temperature for 1 hour, and rabbit TH antibody diluted in TBST (dilution ratio 1:300) was added and left to stand overnight at 4°C. The antibody was then recovered, and the membrane was washed three times with TBST for 10 minutes each time. IRDye diluted in TBST was then added to the membrane. R 680RD goat anti-rabbit (dilution ratio 1:3000) was added, the membrane was washed three times with TBST for 10 min each time, and the protein signals were scanned by a two-color infrared laser imaging system.

[0130] The results of the mouse locomotor activity test are shown in Figure 22. Three to five minutes after MPTP administration, the mice exhibited tremors, decreased movement, arched backs, splayed hind legs, gait instability, vertical tails, and ruffled fur. Individual epileptic seizures occurred approximately 30 to 60 minutes later. The above symptoms gradually subsided, and the mice returned to normal after 24 hours. However, with increasing administration frequency, the acute phase reaction subsided, but only after 24 hours, motor ability decreased, gait instability, and slow response became more evident. After continuous MPTP administration for 7 days, the locomotor distance and movement speed of the mice were significantly lower than those of the blank control group, which showed symptoms of bradykinesia (P<0.01). Administration of AuC alone had no significant effect on the locomotor distance and movement speed of normal mice (Panels A and C of Figure 22). The combined administration of AuC (high dose) to MPTP model mice significantly increased the spontaneous movement distance and movement speed of the mice (Figure 22, Panels B and D), suggesting that AuC plays a significant role in improving the spontaneous movement of MPTP model mice. There was a significant difference compared to the MPTP model group (spontaneous movement distance: P<0.05, movement speed: P<0.01).

[0131] The results of the mouse swimming test are shown in Figure 23. Seven days after continuous MPTP injection, the mice were placed in a water tank and subjected to a swimming test. The longer swimming time and swimming distance of the mice indicate that the mice have better motor coordination of their limbs. The blank control group and the AuC control group did not have a significant effect on the swimming time and swimming distance of the mice (Panels A and C of Figure 23). Compared with the blank control group, the MPTP model group showed a significant decrease in swimming distance in 10 minutes (P<0.05) and a significant decrease in swimming time in the water tank (P<0.05), suggesting that MPTP significantly reduces the swimming motor ability of mice. Compared with the MPTP model group, the AuC (high dose) and AuC-treated groups with MPTP administration showed an increase in swimming distance (P<0.05) and a significant increase in swimming time (P<0.05) (Figure 23, Panels B and D), suggesting that AuC significantly improves MPTP-induced motor behavioral impairment of swimming in mice.

[0132] The results of the rotarod test in mice are shown in Figure 24. After continuous injection of MPTP for 7 days, the mice were subjected to the rotarod test. In the saline-administered blank control group, the latency to fall off the rotarod and the percentage of mice falling off the rotarod were 12.1 ± 4.6 minutes and 33.3 ± 1.5%, respectively (Panels A and C of Figure 24). Compared with the blank control group, the rod fall latency of the MPTP model group mice was significantly shortened to 5.5 ± 3.7 minutes, and the percentage of mice falling off the rod was significantly increased to 83.3 ± 3.4%. After MPTP administration, the mice's motor coordination decreased, and they were unable to firmly grasp the rod and were more likely to fall off the rotarod (Panels B and D of Figure 24). Although administration of AuC alone did not significantly affect the latency to fall (Figure 24, Panel A), the percentage of mice falling off the rotarod during prolonged roller exercise significantly increased (P<0.001, compared to the blank control group). AuC administration itself was shown to have a certain effect on the mice's roller behavior (Figure 24, Panel C). However, compared to the MPTP model group, the AuC-treated group administered both MPTP and AuC showed a significant increase in the latency to fall off the rotarod (low-dose group: P<0.01, high-dose group: P<0.05) and a significant decrease in the percentage of mice falling off the rotarod (P<0.001, for both the high-dose and low-dose groups). The results are shown in Figure 24, Panels B and D. This indicates that AuC has the effect of improving MPTP-induced motor coordination dysfunction.

[0133] The results of immunohistochemical detection in the substantia nigra and striatum and WB detection in the striatum are shown in Figure 25. Compared with the blank control group, the MPTP model group showed a clear decrease in the number of TH-immunopositive neurons (i.e., DAergic neurons) in the substantia nigra, as well as shrinkage and reduced or disappearance of neurites of the remaining neurons, and a decrease in TH-immunopositive neurons and nerve fiber density in the striatum. WB analysis results showed that DAergic neurons in the striatum were reduced to 55.8 ± 5.6% (blank control group: 100%) (P < 0.01 vs. blank control group, see panel C in Figure 25). Administration of AuC alone had no significant effect on the density of TH-immunopositive neurons and nerve fibers in the substantia nigra and striatum (panels A and B in Figure 25). Co-administration of AuC and MPTP could significantly inhibit MPTP-induced downregulation of TH-immunopositive expression in cells and neurofilaments in the substantia nigra and striatum. The results of WB analysis showed that when low-dose AuC was used, the proportion of DAergic neurons in the striatum was 65.6 ± 6.3% of that in the blank control group (P < 0.01, vs. the MPTP model group, see panel C of Figure 25 ), and when high-dose AuC was used, the proportion of DAergic neurons in the striatum reached 84.7 ± 4.5% of that in the blank control group (P < 0.001, vs. the MPTP model group). These results indicate that AuC has a significant effect on resisting MPTP cytotoxicity and exhibits a significant protective effect against DAergic neuron loss in the substantia nigra and striatum.

[0134] The same procedure was also adopted to carry out experiments using AuC modified with various ligands listed in Table 1. Their effects were similar, so they will not be described in detail here.

[0135] The above results demonstrate that the ligand-modified AuC provided in the present invention can significantly improve the locomotor activity, motor ability, and physical coordination ability of MPTP-induced PD model mice, and has a significant protective effect against DAergic neuron loss in the substantia nigra and striatum, indicating that AuC-containing substances can be used for the treatment of PD.

[0136] Embodiment 10: Biosafety Assessment 1. The SH-sy5y cell line was employed to evaluate the biosafety of AuC-containing materials at the cellular level.

[0137] Specific method: SH-sy5y cells (passage 6) in the logarithmic growth phase were harvested. The cell suspension concentration was adjusted and added to each well at 100 μL. The cells were seeded to adjust the cell density to 1,000-10,000 cells per well. The cell culture plate (a 96-well plate with the edge wells filled with cell culture medium) was placed in a cell incubator and incubated at 37°C in 5% CO2 for 24 hours to allow the cells to adhere to the walls. The 96-well plate was then removed, sterilized with alcohol, and placed in a biosafety cabinet. The original cell culture medium was then aspirated, and solutions of the ligand-modified AuC listed in Table 1 were added and diluted with cell culture medium to obtain final concentrations of 1 ppm, 10 ppm, 50 ppm, 100 ppm, 200 ppm, and 500 ppm, respectively. An equal volume of fresh cell culture medium was added to the control group (without AuC). The wells were then placed in a cell incubator and incubated for 48 hours. Six duplicate wells were prepared for each experimental and control group. After 48 hours of incubation, the culture medium was removed by centrifugation and then washed two to three times with PBS. 100 μL of fresh culture medium and 20 μL of methylthiazolyl tetrazolium (MTT) solution (5 mg / ml, i.e., 0.5% MTT) were added to each well, and incubation continued for 4 hours. The incubation was stopped, and the 96-well plate was removed and centrifuged (1,000 rpm) for 10 minutes. The supernatant was aspirated, and 200 μL of DMSO was added to each well. The wells were then placed on a shaking platform and shaken slowly for 10 minutes until the color in the wells became uniform and the crystals were completely dissolved. The absorbance of each well was measured at 490 nm using a microplate reader. The above procedures must be performed in a sterile environment. All steps, except for detection, were completed in a biosafety cabinet. Laboratory supplies must be sterilized in an autoclave before use.

[0138] Taking L-NIBC-modified AuC in Example 2 as an example, the results are shown in Figure 26. Panels A to C show the effect of AuC with particle sizes of 2.6 nm, 1.8 nm, or 1.1 nm on SH-sy5y cell viability at final concentrations of 1 ppm, 10 ppm, 50 ppm, 100 ppm, 200 ppm, or 500 ppm. At fairly high concentrations (e.g., 100 ppm), the addition of L-NIBC-modified AuC had almost no effect on cell viability. At higher concentrations (e.g., 200 ppm and 500 ppm), the addition of L-NIBC-modified AuC resulted in little cell damage (cell death rate of less than 20%). Because 100 ppm is much higher than the minimum effective concentration of AuC (0.1 ppm to 1 ppm or less), it can be concluded that L-NIBC-modified AuC has high safety at the cellular level.

[0139] Other ligand-modified AuCs with various sizes listed in Table 1 also had similar effects, which will not be described in detail here.

[0140] 2. Employing mouse acute toxicity studies to evaluate the acute toxicity of AuC-containing materials Specific Method: For various ligand-modified AuCs listed in Table 1 (taking L-NIBC-modified AuCs with an average diameter of 1.8 nm in Example 2 as an example), 60 adult mice were selected and divided into four groups, each with 15 mice, including one control group and three experimental groups. In the control group, the mice were fed a normal diet, while in the three experimental groups, the mice were orally administered (by gavage) AuCs at doses of 0.1 g / kg body weight, 0.3 g / kg body weight, and 1 g / kg body weight per day, respectively, under normal dietary conditions for one week. After the AuC feeding period ended, the mice were fed a normal diet for 30 days. The mice were observed for any abnormal responses.

[0141] In the mouse experiment described above, the ingestion of three concentrations of AuC with various sizes did not affect the survival and activity of the mice. Even at a high dose of 1 g / kg body weight, the mice remained healthy.

[0142] Other ligand-modified AuCs listed in Table 1 also had similar results, which will not be described in detail here. Based on the above results, it could be concluded that AuC is very safe.

[0143] Embodiment 11: Tissue and metabolic distribution of CuC-containing substances in mice Test 1: Procedure: Eighty mice were randomly divided into four groups with 20 mice in each group, and the ligand-modified AuC listed in Table 1 was orally administered (by gavage) at doses of 100 mg / kg, 20 mg / kg, 5 mg / kg, and 1 mg / kg in the above groups, respectively. After AuC administration, the 20 mice in each group were randomly divided into four subgroups with 5 mice in each subgroup. The mice were sacrificed at 2, 6, 24, and 48 hours after administration. Heart, liver, spleen, lung, kidney, and brain tissues were collected separately. Each tissue was weighed, homogenized in 2 mL of water, then 2 mL of aqua regia was added, vortex-mixed, and shaken on a shaker for 72 hours. 2 wt% nitric acid solution was added to a final volume of 10 mL, and centrifuged at 15,000 rpm for 15 minutes. 4 mL of the supernatant was aspirated, and the content of elemental gold in the tissue fluid was measured by atomic absorption spectroscopy.

[0144] These results showed that AuC could cross the blood-brain barrier and reach the brain. They could be excreted from the body over time, so there was no obvious accumulation in the body. Therefore, the AuC-containing material provided in the present invention has good prospects for use in manufacturing medicines for treating AD or PD.

[0145] Test 2: Procedure: Eighty mice were randomly divided into four groups, with 20 mice in each group, and intraperitoneally injected with the ligand-modified AuC listed in Table 1. The AuC doses in each group (taking L-NIBC-modified AuC with an average diameter of 1.8 nm as an example) were 100 ppm, 20 ppm, 5 ppm, and 1 ppm of mouse body weight, respectively. After AuC injection, the 20 mice in each group were randomly divided into four groups, with five mice in each subgroup. The mice were sacrificed at 2, 6, 24, and 48 hours after feeding. Heart, liver, spleen, lung, kidney, and brain tissues were collected separately. Each tissue was weighed, homogenized in 2 mL of water, and then 2 mL of aqua regia was added, vortex-mixed, and shaken on a shaker for 72 hours. A 2% by weight nitric acid solution was added to make a final volume of 5 mL, and centrifuged at 15000 rpm for 15 minutes. 1 mL of the supernatant was aspirated, and the content of elemental gold in the tissue fluid was measured by atomic absorption spectroscopy.

[0146] The above steps were adopted to perform experiments on AuC modified with other ligands listed in Table 1 .

[0147] The results showed that after 2 hours, the content of elemental gold in the brain reached 1%-10% of the initial concentration. After 6 hours, the content in the brain could be maintained at a similar level. After 24 hours, the content in the brain was significantly reduced. At 48 hours, the content was reduced to near or below the detection limit, except for the sample at a dose of 100 ppm. The results demonstrated that AuC-containing materials have good biosafety at the animal level and can cross the blood-brain barrier. It was shown that there is no obvious accumulation in the body.

[0148] In summary, the above experimental results revealed the following points (all "gold nanoparticles" and "AuC" mentioned below refer to cases with ligand modification):

[0149] (1) In vitro experiments on Aβ aggregation (Embodiment 3) revealed that the effect of gold nanoparticles on Aβ aggregation kinetics is size-related. When the particle size is 10.1 nm or larger, the addition of gold nanoparticles can promote Aβ aggregation. When the particle size is 6.0 nm or smaller, Aβ aggregation is inhibited, but complete inhibition of Aβ aggregation cannot be achieved. However, when AuC is used (average diameter less than 3 nm), all AuC significantly inhibits Aβ aggregation in vitro, and this effect is related to the AuC concentration. When the AuC concentration reaches 5 ppm to 10 ppm, Aβ aggregation can be completely inhibited, and the minimum concentration required for complete inhibition is related to the type of ligand and the diameter of the AuC. In vitro experiments on the inhibition of α-syn aggregation (Embodiment 6) also revealed that AuC has the same effect of completely inhibiting α-syn aggregation and fibrillation.

[0150] (2) Aβ-induced cellular AD model and MPP + In experiments with induced cellular PD models (Embodiments 4, 7, and 8), gold nanoparticles with small particle sizes (e.g., gold nanoparticles with an average diameter of 3.6 nm or 6.0 nm) were found to be effective in the Aβ-induced cellular AD model and MPP. +Since gold nanoparticles were found to have no significant effect on improving cell viability in the induced cellular PD models, this suggests that they do not exhibit any clear therapeutic effect on AD and PD at the cellular level, and therefore cannot be directly used as an active ingredient to manufacture pharmaceuticals for treating AD or PD. However, in the case of various ligand-modified AuC with different sizes (average diameters of less than 3 nm) used in the present invention, even very low doses of AuC (e.g., 0.1 ppm to 1 ppm) were found to be able to increase cell viability in the two models from 50% to 65% to over 95%. At the cellular level, the beneficial effect of AuC was demonstrated to be significant. Because the ligand had no effect on both Aβ aggregation and the two cellular models (Examples 4, 7, and 8), it could be concluded that the beneficial effect of AuC was due to AuC itself. This provided a new approach for the utilization of AuC.

[0151] (3) Furthermore, the transgenic AD mouse model and the MPTP-induced PD mouse model (Embodiment 5 and Embodiment 9) were used in the present invention to further confirm the efficacy of AuC, thus demonstrating that AuC plays a major role in improving the cognitive and motor abilities of mice, inhibiting the formation of senile plaques in the brain, and inhibiting the specific apoptosis of DAergic neurons induced by MPTP in the substantia nigra and striatum, and can be used as a preventive or therapeutic drug for related diseases.

[0152] (4) In an experiment for further evaluation of biosafety (Embodiment 10), when AuC was co-cultured with neurons at a concentration of 100 ppm (by weight), it had no significant effect on cell viability, and when the concentration exceeded 100 ppm (much higher than the minimum effective concentration of AuC), cell viability was slightly reduced. Since the minimum effective concentration of AuC (0.1 ppm to 1 ppm) is much lower than 100 ppm, it can be concluded that AuC has excellent biosafety at the cellular level. In a mouse acute toxicity test, AuC was found to have no adverse effects when administered at a dose of 1 g / kg body weight (equivalent to 1000 ppm) once daily for 7 days. In a study of in vivo distribution and pharmacokinetics in mice (Embodiment 11), the content of elemental gold in the brain reached 1% to 10% of the initial concentration after 2 hours. After 6 hours, the content in the brain remained at a similar level. After 24 hours, the brain contents were significantly reduced. The contents were reduced to below the detection limit except for the specimen at a dose of 100 ppm. The above results indicated that the AuC-containing substance has good biosafety even at the animal level, can pass the blood-brain barrier, and has no obvious accumulation in the body, thus having good prospects for use in the manufacture of medicines for treating AD or PD.

[0153] (5) Compared with current technology, the ligands used in the present invention are not specifically designed for the aggregation behavior of Aβ and α-syn, and comparative experiments have shown that the ligands used have no obvious effect on the aggregation of Aβ and α-syn (Embodiment 3). However, because the size of AuC is smaller than the size of the protein itself, the aggregation of Aβ and α-syn can be largely inhibited by a combination of size effects and weak molecular interactions. The excellent efficacy in Aβ-induced AD cell models and transgenic animal models further confirmed the potential of AuC-containing materials in the manufacture of medicines for the treatment of AD. Furthermore, MPP +The excellent efficacy of AuC-containing substances in the induced PD cell model and the MPTP-induced PD animal model indicates that AuC-containing substances also have a wide application prospect in the manufacture of medicines for treating other neurodegenerative diseases. + The induced PD cell model and the MPTP-induced PD animal model do not involve protein fibrillation but act on deeper mechanisms, including the signal transduction functions related to energy metabolism and neurotransmitter metabolism in neurons. Therefore, it can be speculated that AuC-containing substances may not only affect protein fibrillation but also affect the process of neurodegenerative diseases at a deeper level. AuC-containing substances will be very important for the research and development of new medicines for neurodegenerative diseases. [Industrial Applicability]

[0154] The AuC-containing substance provided in the present invention can improve the cognitive and motor behavioral abilities of mice, inhibit the formation of senile plaques in the brain in an AD transgenic mouse model and an MPTP-induced PD mouse model, and has good biosafety at the animal level. The AuC-containing substance is suitable for industrial use.

Claims

1. AuC and a ligand Y covering the outside of the AuC, the gold core diameter of the AuC is less than 3 nm; The AuC-containing material, wherein the ligand Y is a cysteine-containing tetrapeptide, which is glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) or glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR).

2. 2. The material of claim 1, wherein the gold core diameter of the AuC is between 0.5 nm and 2.6 nm.

3. 3. The material according to claim 1 or 2, wherein the material is in the form of a powder or a floc.

4. A method for producing an AuC-containing material according to any one of claims 1 to 3, comprising: The following steps: (1) HAuCl ４ in one of methanol, water, ethanol, n-propanol, and ethyl acetate to obtain HAuCl ４ obtaining a solution A having a concentration of 0.01 M to 0.03 M; (2) dissolving a ligand Y in a solvent to obtain a solution B having a ligand Y concentration of 0.01 M to 0.18 M; (3) Solution A in step (1) and solution B in step (2) are mixed with HAuCl ４ and ligand Y in a molar ratio of 1:(0.01-100), and the mixture is stirred in an ice bath for 0.1-48 hours, and 0.025-0.8 M NaBH ４ solution was added, and the reaction was then heated in an ice bath for 0.1 to 12 hours with NaBH ４ and ligand Y in a molar ratio of 1:(0.01-100), (4) centrifuging the reaction solution in step (3) at 8,000 rpm to 17,500 rpm for 10 minutes to 100 minutes to obtain AuC precipitates with various average particle sizes; (5) dissolving the AuC precipitates having various average particle sizes obtained in step (4) in water, placing the resulting solution in a dialysis bag, and dialyzing the solution in water at room temperature for 1 to 7 days; (6) freeze-drying the AuC solution in the dialysis bag for 12 to 24 hours to obtain an AuC-containing substance; A method comprising:

5. HAuCl in step (3) ４ The method according to claim 4, wherein the molar ratio between and the ligand Y is 1:(0.1-10).

6. HAuCl in step (3) ４ The method according to claim 4, wherein the molar ratio between and the ligand Y is 1:(1-10).

7. 5. The method according to claim 4, wherein in step (3), the mixture is stirred in an ice bath for 0.1 to 24 hours.

8. 5. The method of claim 4, wherein in step (3), the mixture is stirred in an ice bath for 0.5 to 2 hours.

9. NaBH in step (3) ４ The solution is NaBH ４ an aqueous solution of NaBH ４ or NaBH ４ 5. The method of claim 4, wherein the compound is a methanol solution of

10. NaBH in step (3) ４ The method according to claim 4, wherein the molar ratio of the ligand Y to the ligand is 1:(0.1-8).

11. NaBH in step (3) ４ The method according to claim 4, wherein the molar ratio of the ligand Y to the cation is 1:(1-8).

12. The method according to claim 4, wherein step (4) is a step of obtaining AuCs having various average particle sizes by gradient centrifugation of the reaction solution from step (3) using a tubular ultrafilter having a MWCO of 3K to 30K at 8,000 rpm to 17,500 rpm for 10 minutes to 100 minutes.

13. 13. The method of any one of claims 4 to 12, wherein the solvent in step (2) is one or more of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, and propyl acetate.

14. Use of the AuC-containing material according to any one of claims 1 to 3 in near-infrared fluorescent probes in the fields of catalyst production or molecular catalysis, chiral recognition, molecular detection, biomedical detection, and imaging.

15. Use of the AuC-containing material according to any one of claims 1 to 3 in the manufacture of a medicament for the prevention and / or treatment of a disease associated with Aβ aggregation and fibrillation, and / or α-syn aggregation and fibrillation.

16. Use of the AuC-containing material according to any one of claims 1 to 3 in the manufacture of a medicament for the prevention and / or treatment of Alzheimer's disease (AD).

17. Use of the AuC-containing material according to any one of claims 1 to 3 in the manufacture of a medicament for the prevention and / or treatment of Parkinson's disease (PD).

Images